The present invention relates to a scanner having a confocal optical system, a method for producing focus position data of a confocal optical system of a scanner having a confocal optical system and a method for producing digital data of a scanner having a confocal optical system and, particularly, to a scanner having a confocal optical system, a method for producing focus position data of a confocal optical system of a scanner having a confocal optical system and a method for producing digital data of a scanner having a confocal optical system which can adjust the focus of a confocal optical system with high accuracy without need for special devices and produce digital data for biochemical analysis in a desired manner.
An autoradiography detecting system using as a detecting material for detecting radiation a stimulable phosphor which can absorb, store and record the energy of radiation when it is irradiated with radiation and which, when it is then stimulated by an electromagnetic wave having a specified wavelength, can release stimulated emission whose light amount corresponds to the amount of radiation with which it was irradiated is known, which comprises the steps of introducing a radioactive labeling substance into an organism, using the organism or a part of the tissue of the organism as a specimen, superposing the specimen and a stimulable phosphor sheet formed with a stimulable phosphor layer for a certain period of time, storing and recording radiation energy in a stimulable phosphor contained in the stimulable phosphor layer, scanning the stimulable phosphor layer with an electromagnetic wave to excite the stimulable phosphor, photoelectrically detecting the stimulated emission released from the stimulable phosphor to produce digital image signals, effecting image processing on the obtained digital image signals, and reproducing an image on displaying means such as a CRT or the like or a photographic film (see, for example, Japanese Patent Publication No. 1-60784, Japanese Patent Publication No. 1-60782, Japanese Patent Publication No. 4-3952 and the like).
Unlike the system using a photographic film, according to the autoradiography detecting system using the stimulable phosphor as a detecting material, development, which is chemical processing, becomes unnecessary. Further, it is possible to reproduce a desired image by effecting image processing on the obtained image data and effect quantitative analysis using a computer. Use of a stimulable phosphor in these processes is therefore advantageous.
On the other hand, a fluorescence detecting system using a fluorescent substance as a labeling substance instead of a radioactive labeling substance in the autoradiography detecting-system is known. According to this system, it is possible to study a genetic sequence, to study the expression level of a gene, and to effect separation or identification of protein or estimation of the molecular weight or properties of protein or the like. For example, this system can perform a process including the steps of distributing a plurality of DNA fragments on a gel support by means of electrophoresis after a fluorescent dye was added to a solution containing a plurality of DNA fragments to be distributed, or distributing a plurality of DNA fragments on a gel support containing a fluorescent dye, or dipping a gel support on which a plurality of DNA fragments have been distributed by means of electrophoresis in a solution containing a fluorescent dye, thereby labeling the electrophoresed DNA fragments, exciting the fluorescent dye by a stimulating ray to cause it to release fluorescent light, detecting the released fluorescent light to produce an image and detecting the distribution of the DNA fragments on the gel support. This system can also perform a process including the steps of distributing a plurality of DNA fragments on a gel support by means of electrophoresis, denaturing the DNA fragments, transferring at least a part of the denatured DNA fragments onto a transfer support such as a nitrocellulose support by the Southern-blotting method, hybridizing a probe prepared by labeling target DNA and DNA or RNA complementary thereto with the denatured DNA fragments, thereby selectively labeling only the DNA fragments complementary to the probe DNA or probe RNA, exciting the fluorescent dye by a stimulating ray to cause it to release fluorescent light, detecting the released fluorescent light to produce an image and detecting the distribution of the target DNA on the transfer support. This system can further perform a process including the steps of preparing a DNA probe complementary to DNA containing a target gene labeled by a labeling substance, hybridizing it with DNA on a transfer support, combining an enzyme with the complementary DNA labeled by a labeling substance, causing the enzyme to contact a fluorescent substance, transforming the fluorescent substance to a fluorescent substance having fluorescent light releasing property, exciting the thus produced fluorescent substance by a stimulating ray to release fluorescent light, detecting the fluorescent light to produce an image and detecting the distribution of the target DNA on the transfer support. This fluorescence detecting system is advantageous in that a genetic sequence or the like can be easily detected without using a radioactive substance.
Further, a micro-array detecting system has been recently developed, which comprises the steps of using a spotting device to drop at different positions on the surface of a carrier such as a slide glass plate, a membrane filter or the like specific binding substances, which can specifically bind with a substance derived from a living organism such as a hormone, tumor marker, enzyme, antibody, antigen, abzyme, other protein, a nucleic acid, cDNA, DNA, RNA or the like and whose sequence, base length, composition and the like are known, thereby forming a number of independent spots, specifically binding the specific binding substances using a hybridization method or the like with a substance derived from a living organism such as a hormone, tumor marker, enzyme, antibody, antigen, abzyme, other protein, a nuclear acid, cDNA, DNA or mRNA, which is gathered from a living organism by extraction, isolation or the like or is further subjected to chemical processing, chemical modification or the like and which is labeled with a labeling substance such as a fluorescent substance, dye or the like, thereby forming a micro-array, irradiating the micro-array with a stimulating ray, photoelectrically detecting light such as fluorescence emitted from a labeling substance such as a fluorescent substance, dye or the like, and analyzing the substance derived from a living organism. This micro-array image detecting system is advantageous in that a substance derived from a living organism can be analyzed in a short time period by forming a number of spots of specific binding substances at different positions of the surface of a carrier such as a slide glass plate at high density and hybridizing them with a substance derived from a living organism and labeled with a labeling substance.
In addition, a macro-array detecting system using a radioactive labeling substance as a labeling substance has been further developed, which comprises the steps of using a spotting device to drop at different positions on the surface of a carrier such as a membrane filter or the like specific binding substances, which can specifically bind with a substance derived from a living organism such as a hormone, tumor marker, enzyme, antibody, antigen, abzyme, other protein, a nuclear acid, cDNA, DNA, RNA or the like and whose sequence, base length, composition and the like are known, thereby forming a number of independent spots, specifically binding the specific binding substance using a hybridization method or the like with a substance derived from a living organism such as a hormone, tumor marker, enzyme, antibody, antigen, abzyme, other protein, a nuclear acid, cDNA, DNA or mRNA, which is gathered from a living organism by extraction, isolation or the like or is further subjected to chemical processing, chemical modification or the like and which is labeled with a radioactive labeling substance, thereby forming a macro-array, superposing the macro-array and a stimulable phosphor sheet formed with a stimulable phosphor layer, exposing the stimulable phosphor layer to a radioactive labeling substance, irradiating the stimulable phosphor layer with a stimulating ray to excite the stimulable phosphor, photoelectrically detecting the stimulated emission released from the stimulable phosphor to produce biochemical analysis data, and analyzing the substance derived from a living organism.
These systems are constituted so as to irradiate a sample with a stimulating ray to stimulate a stimulable phosphor or a labeling substance such as a fluorescent substance and photoelectrically detect stimulated emission released from the stimulable phosphor, fluorescence emission released from the fluorescent substance or the like, thereby producing biochemical analysis data such as image data of a labeling substance and emitted light amount data. The data producing apparatuses used in these systems fall in two general categories: those that use a scanner and those that use a two-dimensional sensor.
The data producing apparatus using a scanner is advantageous in that data can be produced with high resolution in comparison with one using a two-dimensional sensor.
The micro-array detecting system is constituted so as to scan a sample with a stimulating ray to stimulate a labeling substance such as a fluorescent substance and photoelectrically detect fluorescence emission released from the fluorescent substance or the like, thereby producing biochemical analysis data such as image data of a labeling substance and emitted light amount data and, therefore, it is indispensable to adjust the focus of a light condensing optical system with high accuracy in order to enable high accurate detection.
Therefore, U.S. Pat. Nos. 5,578,832 and 5,834,785 propose a method for adjusting the focus of a light condensing optical system using an auto-focusing system.
In order to adjust the focus of a light condensing optical system using an auto-focusing system, however, it is necessary to provide special devices such as a reflected light detecting optical system, a sensor, a detection circuit and the like and cost increases in proportion.
It is therefore an object of the present invention to provide a scanner having a confocal optical system, a method for producing focus position data of a confocal optical system of a scanner having a confocal optical system and a method for producing digital data of a scanner having a confocal optical system which can adjusting the focus of a confocal optical system with high accuracy without need for special devices and produce digital data for biochemical analysis in a desired manner.
The above and other objects of the present invention can be accomplished by a scanner comprising at least one laser stimulating ray source for emitting a laser beam, a sample stage on which a sample carrier for carrying at least one sample is to be placed, a scanning means for moving the sample stage in a main scanning direction and in a sub-scanning direction, a confocal optical system, a drive means for an objective lens incorporated in the confocal optical system, a light detector for photoelectrically detecting light, a non-volatile memory, and a control means, the non-volatile memory being constituted so as to store position data produced by setting at least one distance measuring device in the sample carrier for carrying at least one sample, placing the sample carrier on the sample stage, and measuring a distance between the objective lens incorporated in the confocal optical system and a reference position on a surface of the at least one distance measuring device set in the sample carrier and a distance between the objective lens and at least one measurement position on a surface of the at least one distance measuring device different from the reference position, and to store focus position data produced by setting a focus position determination device including a luminescent material having a property to release fluorescence emission or photoluminescence emission upon being irradiated with the laser beam in the sample carrier so that the luminescent material is located at the reference position, scanning the focus position determination device with the laser beam to stimulate the luminescent material located at the reference position, photoelectrically detecting fluorescence emission or photoluminescence emission released from the luminescent material by the light detector, changing the position of the objective lens of the confocal optical system with a predetermined pitch, and determining a focus position of the confocal optical system, the control means being constituted so as to correct the focus position data of the confocal optical system stored in the non-volatile memory with the position data stored in the non-volatile memory, and output a drive signal to the drive means based on the thus corrected focus position data of the confocal optical system, thereby causing it to move the objective lens of the confocal optical system and adjust the position of the objective lens.
According to the present invention, the non-volatile memory is constituted so as to store position data produced by setting at least one distance measuring device in the sample carrier for carrying at least one sample, placing the sample carrier on the sample stage, and measuring the distance between the objective lens incorporated in the confocal optical system and a reference position on the surface of the at least one distance measuring device set in the sample carrier and the distance between the objective lens and at least one measurement position on the surface of the at least one distance measuring device different from the reference position, and to store focus position data produced by setting a focus position determination device including a luminescent material having a property to release fluorescence emission or photoluminescence emission upon being irradiated with the laser beam in the sample carrier so that the luminescent material is located at the reference position, scanning the focus position determination device with the laser beam to stimulate the luminescent material located at the reference position, photoelectrically detecting fluorescence emission or photoluminescence emission released from the luminescent material by the light detector, changing the position of the objective lens of the confocal optical system with a predetermined pitch, and determining the focus position of the confocal optical system, and the control means is constituted so as to correct the focus position data of the confocal optical system stored in the non-volatile memory with the position data stored in the non-volatile memory, and output a drive signal to the drive means based on the thus corrected focus position data of the confocal optical system, thereby causing it to move the objective lens of the confocal optical system and adjust the position of the objective lens. Therefore, since the position of the objective lens of the confocal optical system can be adjusted based on the distance between the at least one distance determination device set in the sample carrier and the objective lens of the confocal optical system, the focus of the confocal optical system can be adjusted in a desired manner without an auto-focusing system provided with special devices such as a reflected light detecting optical system, a sensor, a detection circuit and the like.
In a preferred aspect of the present invention, the position of the objective lens of the confocal optical system at which an integrated value of signal intensity of fluorescence emission or photoluminescence emission detected by the light detector becomes maximum is determined as the focus position of the confocal optical system and is stored in the non-volatile memory as the focus position data.
In a further preferred aspect of the present invention, the position data are produced by measuring the distance between the objective lens incorporated in the confocal optical system and the reference position on the surface of the at least one distance measuring device set in the sample carrier and the distance between the objective lens and the at least one measurement position on the surface of the at least one distance measuring device different from the reference position and calculating a displacement of the at least one measurement position on the surface of the at least one distance measuring device set in the sample carrier different from the reference position with respect to the reference position on the surface of the at least one distance measuring device and are stored in the non-volatile memory.
In a further preferred aspect of the present invention, the position data are produced by measuring the distance between the objective lens incorporated in the confocal optical system and the reference position on the surface of the at least one distance measuring device set in the sample carrier and the distance between the objective lens and two or more measurement positions different from the reference position on the surface of the at least one distance measuring device, calculating displacements of the two or more measurement positions different from the reference position on the surface of the at least one distance measuring device set in the sample carrier with respect to the reference position on the surface of the at least one distance measuring device, and averaging the displacements of the two or more measurement positions different from the reference position on the surface of the at least one distance measuring device with respect to the reference position, and are stored in the non-volatile memory.
According to this preferred aspect of the present invention, it is possible to more accurately adjust the focus of the confocal system because the position data are produced by measuring the distance between the objective lens incorporated in the confocal optical system and the reference position on the surface of the at least one distance measuring device set in the sample carrier and the distance between the objective lens and two or more measurement positions different from the reference position on the surface of the at least one distance measuring device, calculating displacements of the two or more measurement positions different from the reference position on the surface of the at least one distance measuring device set in the sample carrier with respect to the reference position on the surface of the at least one distance measuring device, and averaging the displacements of the two or more measurement positions different from the reference position on the surface of the at least one distance measuring device with respect to the reference position, and are stored in the non-volatile memory.
In a further preferred aspect of the present invention, the scanner further comprises a temperature sensor for detecting a temperature in the scanner and the non-volatile memory is constituted so as to store the temperature coefficients of the displacements of the two or more measurement positions different from the reference position on the surface of the at least one distance measuring device with respect to the reference position produced by measuring the distance between the objective lens incorporated in the confocal optical system and the reference position on the surface of the at least one distance measuring device set in the sample carrier and the distance between the objective lens and two or more measurement positions different from the reference position on the surface of the at least one distance measuring device at two or more different temperatures from each other, and calculating displacements of the two or more measurement positions different from the reference position on the surface of the at least one distance measuring device set in the sample carrier with respect to the reference position on the surface of the at least one distance measuring device, and to store an average value of temperatures in the scanner detected by the temperature sensor when the focus position data of the confocal optical system were produced, and the control means is constituted so as to correct the focus position data of the confocal optical system stored in the non-volatile memory according to difference in temperature between a temperature in the scanner detected by the temperature sensor and the average value of temperatures in the scanner when the focus position data of the confocal optical system were produced and in accordance with the temperature coefficients of the two or more measurement positions different from the reference position on the surface of each of the two or more distance measuring devices with respect to the reference position stored in the non-volatile memory.
According to this preferred aspect of the present invention, it is possible to more accurately adjust the focus of the confocal system even when the temperature in the scanner changes because the non-volatile memory is constituted so as to store the temperature coefficients of the displacements of the two or more measurement positions different from the reference position on the surface of the at least one distance measuring device with respect to the reference position produced by measuring the distance between the objective lens incorporated in the confocal optical system and the reference position on the surface of the at least one distance measuring device set in the sample carrier and the distance between the objective lens and two or more measurement positions different from the reference position on the surface of the at least one distance measuring device at two or more different temperatures from each other, and calculating displacements of the two or more measurement positions different from the reference position on the surface of the at least one distance measuring device set in the sample carrier with respect to the reference position on the surface of the at least one distance measuring device, and to store an average value of temperatures in the scanner detected by the temperature sensor when the focus position data of the confocal optical system were produced, and the control means is constituted so as to correct the focus position data of the confocal optical system stored in the non-volatile memory according to difference in temperature between a temperature in the scanner detected by the temperature sensor and the average value of temperatures in the scanner when the focus position data of the confocal optical system were produced and in accordance with the temperature coefficients of the displacements of the two or more measurement positions different from the reference position on the surface of the at least one distance measuring device with respect to the reference position stored in the non-volatile memory.
In a further preferred aspect of the present invention, the sample carrier is constituted so as to carry two or more distance measuring devices, the non-volatile memory is constituted so as to store the position data produced by placing the sample carrier carrying two or more samples on the sample stage, measuring distances between the objective lens incorporated in the confocal optical system and three or more measurement positions different from each other on each of the two or more distance measuring devices, determining one of the measurement positions on one of the two or more distance measuring devices as the reference position, calculating displacements of the measurement positions on each of the two or more distance measuring devices with respect to the reference position, and averaging the displacements of the measurement positions on each of the two or more distance measuring devices, and the control means is constituted so as to correct the focus position data of the confocal optical system stored in the non-volatile memory based on the position data stored in the non-volatile memory for each of samples set at positions corresponding to positions of the two or more distance measuring devices set in the sample carrier, and output a drive signal to the drive means in accordance with the thus corrected focus position data of the confocal optical system, thereby causing it to move the objective lens of the confocal optical system and adjust the position of the objective lens.
According to this preferred aspect of the present invention, it is possible to accurately adjust the focus of the confocal system for each of a plurality of samples set in the sample carrier because the sample carrier is constituted so as to carry two or more distance measuring devices, the non-volatile memory is constituted so as to store the position data produced by placing the sample carrier carrying two or more samples on the sample stage, measuring distances between the objective lens incorporated in the confocal optical system and three or more measurement positions different from each other on each of the two or more distance measuring devices, determining one of the measurement positions on one of the two or more distance measuring devices as the reference position, calculating displacements of the measurement positions on each of the two or more distance measuring devices with respect to the reference position, and averaging the displacements of the measurement positions on each of the two or more distance measuring devices, and the control means is constituted so as to correct the focus position data of the confocal optical system stored in the non-volatile memory based on the position data stored in the non-volatile memory for each of samples set at positions corresponding to positions of the two or more distance measuring devices set in the sample carrier, and output a drive signal to the drive means in accordance with the thus corrected focus position data of the confocal optical system, thereby causing it to move the objective lens of the confocal optical system and adjust the position of the objective lens.
In a further preferred aspect of the present invention, the temperature coefficients of displacements of the measurement positions with respect to the reference position are calculated by measuring distances between the objective lens incorporated in the confocal optical system and the three or more measurement positions on each of the two or more distance measuring devices determined by one of the measurement positions on one of the two or more distance measuring devices at two or more temperatures different from each other, determining one of the measurement positions on one of the two or more distance measuring devices as the reference position and calculating displacements of the measurement positions on each of the two or more distance measuring devices with respect to the reference position, the non-volatile memory is constituted so as to store an average value of the temperature coefficients of displacements of the measurement positions with respect to the reference position as the temperature coefficient of each of the two or more distance measuring devices and to store an average value of the temperatures in the scanner detected by the temperature sensor when the focus position data were produced, and the control means is constituted so as to correct the focus position data of the confocal optical system stored in the non-volatile memory for each of the two or more distance measuring devices according to difference in temperature between a temperature in the scanner detected by the temperature sensor and the average value of temperatures in the scanner when the focus position data of the confocal optical system were produced and in accordance with the temperature coefficients of the two or more measurement positions different from the reference position on the surface of each of the two or more distance measuring devices with respect to the reference position stored in the non-volatile memory, and output a drive signal to the drive means in accordance with the thus corrected focus position data of the confocal optical system, thereby causing it to move the objective lens of the confocal optical system and adjust the position of the objective lens.
According to this preferred aspect of the present invention, it is possible to accurately adjust the focus of the confocal system for each of a plurality of samples set in the sample carrier even when the temperature in the scanner changes because the temperature coefficients of displacements of the measurement positions with respect to the reference position are calculated by measuring distances between the objective lens incorporated in the confocal optical system and the three or more measurement positions on each of the two or more distance measuring devices at two or more temperatures different from each other and calculating displacements of the measurement positions on each of the two or more distance measuring devices with respect to the reference position, the non-volatile memory is constituted so as to store an average value of the temperature coefficients of displacements of the measurement positions with respect to the reference position as the temperature coefficient of each of the two or more distance measuring devices and to store an average value of the temperatures in the scanner detected by the temperature sensor when the focus position data were produced, and the control means is constituted so as to correct the focus position data of the confocal optical system stored in the non-volatile memory for each of the two or more distance measuring devices according to difference in temperature between a temperature in the scanner detected by the temperature sensor and the average value of temperatures in the scanner when the focus position data of the confocal optical system were produced and in accordance with the temperature coefficients of the two or more distance measuring devices stored in the non-volatile memory, and output a drive signal to the drive means in accordance with the thus corrected focus position data of the confocal optical system, thereby causing it to move the objective lens of the confocal optical system and adjust the position of the objective lens.
In a further preferred aspect of the present invention, the non-volatile memory is constituted so as to store the position data produced by measuring distances between the objective lens incorporated in the confocal optical system and at least two measurement positions different from each other in each main scanning line on each of the two or more distance measuring devices set in the sample carrier, determining one of the measurement positions on one of the two or more distance measuring devices as the reference position, calculating displacements of the measurement positions on each of the two or more distance measuring devices with respect to the reference position, and averaging the displacements of the measurement positions on each of the two or more distance measuring devices.
In a further preferred aspect of the present invention, the temperature coefficients of displacements of the measurement positions with respect to the reference position are calculated by measuring distances between the objective lens incorporated in the confocal optical system and the at least two measurement positions in each main scanning line on each of the two or more distance measuring devices at two or more temperatures different from each other, determining one of the measurement positions on one of the two or more distance measuring devices as the reference position and calculating displacements of the measurement positions on each of the two or more distance measuring devices with respect to the reference position, the non-volatile memory is constituted so as to store an average value of the temperature coefficients of displacements of the measurement positions with respect to the reference position as the temperature coefficient of each of the two or more distance measuring devices and to store an average value of the temperatures in the scanner detected by the temperature sensor when the focus position data were produced, and the control means is constituted so as to correct the focus position data of the confocal optical system stored in the non-volatile memory for each of the two or more distance measuring devices according to difference in temperature between a temperature in the scanner detected by the temperature sensor and the average value of temperatures in the scanner when the focus position data of the confocal optical system were produced and in accordance with the temperature coefficients of the two or more distance measuring devices stored in the non-volatile memory, and output a drive signal to the drive means in accordance with the thus corrected focus position data of the confocal optical system, thereby causing it to move the objective lens of the confocal optical system and adjust the position of the objective lens.
According to this preferred aspect of the present invention, it is possible to more accurately adjust the focus of the confocal system for each of a plurality of samples set in the sample carrier even when the temperature in the scanner changes because the temperature coefficients of displacements of the measurement positions with respect to the reference position are calculated by measuring distances between the objective lens incorporated in the confocal optical system and the at least two measurement positions in each main scanning line on each of the two or more distance measuring devices at two or more temperatures different from each other, determining one of the measurement positions on one of the two or more distance measuring devices as the reference position and calculating displacements of the measurement positions on each of the two or more distance measuring devices with respect to the reference position, the non-volatile memory is constituted so as to store an average value of the temperature coefficients of displacements of the measurement positions with respect to the reference position as the temperature coefficient of each of the two or more distance measuring devices and to store an average value of the temperatures in the scanner detected by the temperature sensor when the focus position data were produced, and the control means is constituted so as to correct the focus position data of the confocal optical system stored in the non-volatile memory for each of the two or more distance measuring devices according to difference in temperature between a temperature in the scanner detected by the temperature sensor and the average value of temperatures in the scanner when the focus position data of the confocal optical system were produced and in accordance with the temperature coefficients of the two or more distance measuring devices stored in the non-volatile memory, and output a drive signal to the drive means in accordance with the thus corrected focus position data of the confocal optical system, thereby causing it to move the objective lens of the confocal optical system and adjust the position of the objective lens.
In a preferred aspect of the present invention, the non-volatile memory is constituted so as to store the position data produced by measuring distances between the objective lens incorporated in the confocal optical system and three measurement positions different from each other in each main scanning line on each of the two or more distance measuring devices set in the sample carrier, determining one of the measurement positions on one of the two or more distance measuring devices as the reference position, calculating displacements of the measurement positions on each of the two or more distance measuring devices with respect to the reference position, and averaging the displacements of the measurement positions on each of the two or more distance measuring devices.
In a further preferred aspect of the present invention, the temperature coefficients of displacements of the measurement positions with respect to the reference position are calculated by measuring distances between the objective lens incorporated in the confocal optical system and the three measurement positions in each main scanning line on each of the two or more distance measuring devices at two or more temperatures different from each other, determining one of the measurement positions on one of the two or more distance measuring devices as the reference position and calculating displacements of the measurement positions on each of the two or more distance measuring devices with respect to the reference position, the non-volatile memory is constituted so as to store an average value of the temperature coefficients of displacements of the measurement positions with respect to the reference position as the temperature coefficient of each of the two or more distance measuring devices and to store an average value of the temperatures in the scanner detected by the temperature sensor when the focus position data were produced, and the control means is constituted so as to correct the focus position data of the confocal optical system stored in the non-volatile memory for each of the two or more distance measuring devices according to difference in temperature between a temperature in the scanner detected by the temperature sensor and the average value of temperatures in the scanner when the focus position data of the confocal optical system were produced and in accordance with the temperature coefficients of the two or more distance measuring devices stored in the non-volatile memory, and output a drive signal to the drive means in accordance with the thus corrected focus position data of the confocal optical system, thereby causing it to move the objective lens of the confocal optical system and adjust the position of the objective lens.
In a further preferred aspect of the present invention, the non-volatile memory is constituted so as to store the position data produced by averaging the displacements of the measurement positions with respect to the reference position for each predetermined number of main scanning lines on each of the two or more distance measuring devices, and the control means is constituted so as to correct the focus position data of the confocal optical system stored in the non-volatile memory for each number of main scanning lines corresponding to the predetermined number of main scanning lines of the samples set at positions in the sample carrier corresponding to positions where the two or more distance measuring devices were set based on the position data stored in the non-volatile memory, and output a drive signal to the drive means in accordance with the thus corrected focus position data of the confocal optical system, thereby causing it to move the objective lens of the confocal optical system and adjust the position of the objective lens.
According to this preferred aspect of the present invention, the non-volatile memory is constituted so as to store the position data produced by averaging the displacements of the measurement positions with respect to the reference position for each predetermined number of main scanning lines on each of the two or more distance measuring devices, and the control means is constituted so as to correct the focus position data of the confocal optical system stored in the non-volatile memory for each number of main scanning lines corresponding to the predetermined number of main scanning lines of the samples set at positions in the sample carrier corresponding to positions where the two or more distance measuring devices were set based on the position data stored in the non-volatile memory, and output a drive signal to the drive means in accordance with the thus corrected focus position data of the confocal optical system, thereby causing it to move the objective lens of the confocal optical system and adjust the position of the objective lens, and, therefore, the position of the objective lens can be more precisely adjusted to enable more accurate adjustment of the focus of the confocal system for each of the plurality of samples set in the sample carrier.
In a further preferred aspect of the present invention, the temperature coefficients of displacements of the measurement positions with respect to the reference position are calculated by measuring distances between the objective lens incorporated in the confocal optical system and three measurement positions in each main scanning line for each predetermined number of main scanning lines on each of the two or more distance measuring devices at two or more temperatures different from each other, determining one of the measurement positions on one of the two or more distance measuring devices as the reference position and calculating displacements of the measurement positions for each predetermined number of main scanning lines on each of the two or more distance measuring devices with respect to the reference position, the non-volatile memory is constituted so as to store an average value of the temperature coefficients of displacements of the measurement positions with respect to the reference position for each predetermined number of main scanning lines as the temperature coefficient of each predetermined number of main scanning lines of each of the two or more distance measuring devices and to store an average value of the temperatures in the scanner detected by the temperature sensor when the focus position data were produced, and the control means is constituted so as to correct the focus position data of the confocal optical system stored in the non-volatile memory for each number of main scanning lines corresponding to the predetermined number of main scanning lines of the samples set at positions in the sample carrier corresponding to positions where the two or more distance measuring devices were set according to difference in temperature between a temperature in the scanner detected by the temperature sensor and the average value of temperatures in the scanner when the focus position data of the confocal optical system were produced and in accordance with the temperature coefficients of the predetermined number of main scanning lines on each of the two or more distance measuring devices, and output a drive signal to the drive means in accordance with the thus corrected focus position data of the confocal optical system, thereby causing it to move the objective lens of the confocal optical system and adjust the position of the objective lens.
According to this preferred aspect of the present invention, the temperature coefficients of displacements of the measurement positions with respect to the reference position are calculated by measuring distances between the objective lens incorporated in the confocal optical system and three measurement positions in each main scanning line for each predetermined number of main scanning lines on each of the two or more distance measuring devices at two or more temperatures different from each other, determining one of the measurement positions on one of the two or more distance measuring devices as the reference position and calculating displacements of the measurement positions for each predetermined number of main scanning lines on each of the two or more distance measuring devices with respect to the reference position, the non-volatile memory is constituted so as to store an average value of the temperature coefficients of displacements of the measurement positions with respect to the reference position for each predetermined number of main scanning lines as the temperature coefficient of each predetermined number of main scanning lines of each of the two or more distance measuring devices and to store an average value of the temperatures in the scanner detected by the temperature sensor when the focus position data were produced, and the control means is constituted so as to correct the focus position data of the confocal optical system stored in the non-volatile memory for each number of main scanning lines corresponding to the predetermined number of main scanning lines of the samples set at positions in the sample carrier corresponding to positions where the two or more distance measuring devices were set according to difference in temperature between a temperature in the scanner detected by the temperature sensor and the average value of temperatures in the scanner when the focus position data of the confocal optical system were produced and in accordance with the temperature coefficients of the predetermined number of main scanning lines on each of the two or more distance measuring devices, and output a drive signal to the drive means in accordance with the thus corrected focus position data of the confocal optical system, thereby causing it to move the objective lens of the confocal optical system and adjust the position of the objective lens, and therefore the position of the objective lens can be more precisely adjusted even when the temperature in the scanner changes to enable more accurate adjustment of the focus of the confocal system for each of the plurality of samples set in the sample carrier.
In a further preferred aspect of the present invention, the scanner further comprises two or more laser stimulating ray sources for emitting laser beams having different wavelengths from each other and the non-volatile memory is constituted so as to store the focus position data of the confocal optical system produced for each wavelength of the laser beam, and the control means is constituted so as to read from the non-volatile memory the focus position data of the confocal optical system corresponding to the wavelength of the laser beam emitted from the laser stimulating ray source to be used for scanning the at least one sample from among the two or more laser stimulating ray sources, and output a drive signal to the drive means in accordance with the thus read the focus position data of the confocal optical system, thereby causing it to move the objective lens of the confocal optical system and adjust the position of the objective lens.
Although the focus position of the confocal optical system depends upon the wavelength of the laser beam, i.e., the stimulating ray, according to this preferred aspect of the present invention, the focus position of the confocal optical system can be adjusted in a desired manner when the laser beam having a wavelength capable of most efficiently stimulating a labeling substance, a fluorescent dye, is selected to stimulate a sample(s) and fluorescence emission released from the sample is photoelectrically detected, because the non-volatile memory is constituted so as to store the focus position data of the confocal optical system produced for each wavelength of the laser beam, and the control means is constituted so as to read from the non-volatile memory the focus position data of the confocal optical system corresponding to the wavelength of the laser beam emitted from the laser stimulating ray source to be used for scanning the at least one sample from among the two or more laser stimulating ray sources, and output a drive signal to the drive means in accordance with the thus read the focus position data of the confocal optical system, thereby causing it to move the objective lens of the confocal optical system and adjust the position of the objective lens.
In a further preferred aspect of the present invention, the distance measuring device is provided with a metal layer on the surface thereof and the position data are produced using an electrostatic capacitance displacement meter.
According to this preferred aspect of the present invention, since the distance measuring device is provided with a metal layer on the surface thereof and the position data are produced using an electrostatic capacitance displacement meter, it is possible to produce the position data on the sub-micron order to enable accurate adjustment of the focus position of the confocal optical system.
In a further preferred aspect of the present invention, the position data are optically produced.
According to this preferred aspect of the present invention, in the case where the position data are produced using an auto-focusing system, a laser displacement meter or the like and the thus produced position data are stored in the non-volatile memory, the scanner itself does not require auto-focusing capability, and, therefore, it is possible to adjust the focus position of the confocal optical system in a desired manner without need for an auto-focusing system including special devices such as a reflected light detection system, a sensor, a detection circuit and the like.
In a further preferred aspect of the present invention, the metal layer of the distance measuring device is formed by a method selected from a group consisting of sputtering, chemical vapor deposition and evaporation.
In a further preferred aspect of the present invention, the metal layer of the distance measuring device is formed by sputtering.
In a further preferred aspect of the present invention, the metal layer of the distance measuring device is formed of a material selected from a group consisting of chromium, aluminum, gold, nickel-chromium alloy and titanium-nickel-chromium alloy.
In a further preferred aspect of the present invention, the metal layer of the distance measuring device is formed of chromium.
In a further preferred aspect of the present invention, the focus position determination device is constituted as a slide glass plate including a spot containing at least one kind of fluorescent dye formed on the surface thereof.
In a further preferred aspect of the present invention, the spot contains two or more kinds of fluorescent dyes capable of efficiently being stimulated by laser beams having different wavelengths from each other.
Although the focus position of the confocal optical system depends upon the wavelength of the laser beam, i.e., the stimulating ray, according to this preferred aspect of the present invention, since the spot contains two or more kinds of fluorescent dyes capable of efficiently being stimulated by laser beams having different wavelengths from each other, it is possible to produce the focus position data of the confocal optical system for each wavelength of the laser beam and store them in the non-volatilememory and, therefore, possible to adjust the focus position of the confocal optical system in a desired manner when the laser beam having a wavelength capable of most efficiently stimulating a labeling substance, a fluorescent dye, is selected to stimulate a sample(s) and fluorescence emission released from the sample is photoelectrically detected.
In a further preferred aspect of the present invention, the spot contains a fluorescent dye capable of being efficiently stimulated by a laser beam having a wavelength between 470 nm and 490 nm, a fluorescent dye capable of being efficiently stimulated by a laser beam having a wavelength between 530 nm and 540 nm and a fluorescent dye capable of being efficiently stimulated by a laser beam having a wavelength between 630 nm and 650 nm.
Although the focus position of the confocal optical system depends upon the wavelength of the laser beam, i.e., the stimulating ray, according to this preferred aspect of the present invention, the focus position of the confocal optical system can be adjusted in a desired manner when the laser beam having a wavelength capable of most efficiently stimulating a labeling substance, a fluorescent dye, is selected to stimulate a sample(s) and fluorescence emission released from the sample is photoelectrically detected, because the focus position data of the confocal optical system can be produced for a laser beam having a wavelength between 470 nm and 490 nm, a laser beam having a wavelength between 530 nm and 540 nm and having a wavelength between 630 nm and 650 nm using the spot containing a fluorescent dye capable of being efficiently stimulated by a laser beam having a wavelength between 470 nm and 490 nm, a fluorescent dye capable of being efficiently stimulated by a laser beam having a wavelength between 530 nm and 540 nm and a fluorescent dye capable of being efficiently stimulated by a laser beam having a wavelength between 630 nm and 650 nm and stored in the non-volatile memory.
In a further preferred aspect of the present invention, the focus position determination device is formed by providing the metal layer on a support formed of an inorganic material which can release fluorescence emission or photoluminescence emission and is not degraded by the irradiation with the laser beam so as to form an opening through which the support is exposed.
Although a fluorescent dye is sometimes degraded with the lapse of time when being irradiated with a laser beam, whereby the amount of released fluorescence emission is lowered and the focus position of the confocal optical system cannot be determined in a desired manner, according to this preferred aspect of the present invention, since the focus position determination device is formed by providing the metal layer on a support formed of an inorganic material which can release fluorescence emission or photoluminescence emission and is not degraded by the irradiation with the laser beam so as to form an opening through which the support is exposed, it is possible to accurately determine the focus position of the confocal optical system in a desired manner.
In a further preferred aspect of the present invention, the support is formed of a material selected from a group consisting of IV group elements, II-VI group compounds, III-V group compounds and complexes thereof.
In a further preferred aspect of the present invention, the support is formed by a color glass filter formed by doping glass containing a material selected from a group consisting of silica sand, soda ash and limestone as a principal component with a solid solution of CdSxe2x80x94CdSe.
Although a fluorescent dye is sometimes degraded with the lapse of time when being irradiated with a laser beam, whereby the amount of released fluorescence emission is lowered and the focus position of the confocal optical system cannot be determined in a desired manner, according to this preferred aspect of the present invention, since the support is formed by a color glass filter formed by doping glass containing a material selected from a group consisting of silica sand, soda ash and limestone as a principal component with a solid solution of CdSxe2x80x94CdSe and is not degraded by the irradiation with the laser beam, it is possible to accurately determine the focus position of the confocal optical system in a desired manner.
In a further preferred aspect of the present invention, the support is formed by a color glass filter formed by doping glass containing a material selected from a group consisting of silica sand, soda ash and limestone as a principal component with a solid solution of ZnSxe2x80x94CdS.
Although a fluorescent dye is sometimes degraded with the lapse of time when being irradiated with a laser beam, whereby an amount of released fluorescence emission is lowered and the focus position of the confocal optical system cannot be determined in a desired manner, according to this preferred aspect of the present invention, since the support is formed by a color glass filter formed by doping glass containing a material selected from a group consisting of silica sand, soda ash and limestone as a principal component with a solid solution of ZnSxe2x80x94CdS and is not degraded by the irradiation with the laser beam, it is possible to accurately determine the focus position of the confocal optical system in a desired manner.
In a further preferred aspect of the present invention, the support is formed by a laminate of an InGaAsP layer and a GaAs layer and the metal layer is formed on the InGaAsP layer.
Although a fluorescent dye is sometimes degraded with the lapse of time when being irradiated with a laser beam, whereby an amount of released fluorescence emission is lowered and the focus position of the confocal optical system cannot be determined in a desired manner, according to this preferred aspect of the present invention, since the support is formed by a laminate of an InGaAsP layer and a GaAs layer, which are not degraded by the irradiation with the laser beam, and the metal layer is formed on the InGaAsP layer, it is possible to accurately determine the focus position of the confocal optical system in a desired manner.
In a further preferred aspect of the present invention, the metal layer is formed by a method selected from a group consisting of sputtering, chemical vapor deposition and evaporation.
In a further preferred aspect of the present invention, the metal layer is formed by sputtering.
In a further preferred aspect of the present invention, the metal layer is formed of a material selected from a group consisting of chromium, aluminum, gold, nickel-chromium alloy and titanium-nickel-chromium alloy.
In a further preferred aspect of the present invention, the metal layer is formed of chromium.
In a further preferred aspect of the present invention, the drive means is constituted as a stepping motor.
In a further preferred aspect of the present invention, the non-volatile memory is constituted so as to store the focus position data in the form of drive pulses applied to the stepping motor and the control means is constituted so as to correct the number of drive pulses stored in the non-volatile memory, and apply the thus corrected number of drive pulses to the stepping motor, thereby causing it to move the objective lens of the confocal optical system and adjust the position of the objective lens.
In a further preferred aspect of the present invention, the scanner further comprises a data processing apparatus, and the non-volatile memory is constituted so as to store coefficients of an nth order function produced by plotting integrated values of signal intensity of fluorescence emission or photoluminescence emission detected by the light detector and fitting the plotted values with the nth order function by the control means and displacements of measurement positions on the at least one distance measuring device with respect to the reference position, the control means or the data processing apparatus is constituted so as to produce shading correction data for correcting shading of digital data of the at least one sample based on the coefficients of the nth order function and the displacements of measurement positions on the at least one distance measuring device with respect to the reference position stored in the non-volatile memory, and the data processing apparatus is constituted so as to correct the digital data of the at least one sample based on the shading correction data.
According to this preferred aspect of the present invention, it is possible to produce shading correction data as well as the focus position data of the confocal optical system and produce the digital data of at least one sample in which the shading has been corrected, because the non-volatile memory is constituted so as to store coefficients of an nth order function produced by plotting integrated values of signal intensity of fluorescence emission or photoluminescence emission detected by the light detector and fitting the plotted values with the nth order function by the control means and displacements of measurement positions on the at least one distance measuring device with respect to the reference position, the control means or the data processing apparatus is constituted so as to produce shading correction data for correcting shading of digital data of the at least one sample based on the coefficients of the nth order function and the displacements of measurement positions on the at least one distance measuring device with respect to the reference position stored in the non-volatile memory, and the data processing apparatus is constituted so as to correct the digital data of the at least one sample based on the shading correction data.
In a further preferred aspect of the present invention, the scanner further comprises a data processing apparatus, and the non-volatile memory is constituted so as to store shading correction data for correcting shading of digital data of the at least one sample produced based on coefficients of an nth order function produced by plotting integrated values of signal intensity of fluorescence emission or photoluminescence emission detected by the light detector and fitting the plotted values with the nth order function by the control means and displacements of measurement positions on the at least one distance measuring device with respect to the reference position, and the data processing apparatus is constituted so as to correct the digital data of the at least one sample based on the shading correction data.
According to this preferred aspect of the present invention, it is possible to produce shading correction data as well as the focus position data of the confocal optical system and produce the digital data of the sample in which the shading has been corrected, because the non-volatile memory is constituted so as to store shading correction data for correcting shading of digital data of the at least one sample produced based on coefficients of an nth order function produced by plotting integrated values of signal intensity of fluorescence emission or photoluminescence emission detected by the light detector and fitting the plotted values with the nth order function by the control means and displacements of measurement positions on the at least one distance measuring device with respect to the reference position, and the data processing apparatus is constituted so as to correct the digital data of the at least one sample based on the shading correction data.
In a further preferred aspect of the present invention, the scanner further comprises a data processing apparatus provided with a memory, the data processing apparatus is constituted so as to produce shading correction data based on digital data of a shading estimation device produced by setting the shading estimation device in which a mask of metal is formed on a support having a property to release fluorescence emission upon being irradiated with a laser beam and capable being processed while retaining optical flatness, thereby regularly forming a plurality of openings through which the support is exposed in the sample carrier, placing the sample carrier on the sample stage, scanning the shading estimation device with the laser beam emitted from the at least one laser stimulating ray source via the openings to stimulate the support at the plurality of openings, photoelectrically detecting fluorescence emission released from the support via the plurality of openings by the light detector to produce analog data, digitizing the analog data, to store the shading correction data of the shading estimation device in the non-volatile memory or the memory of the data processing apparatus, and to correct digital data of the at least one sample based on the shading correction data stored in the non-volatile memory or the memory of the data processing apparatus.
According to this preferred aspect of the present invention, the scanner further comprises a data processing apparatus provided with a memory, the data processing apparatus is constituted so as to produce shading correction data based on digital data of a shading estimation device produced by setting the shading estimation device in which a mask of metal is formed on a support having a property to release fluorescence emission upon being irradiated with a laser beam and capable of being processed while retaining optical flatness, thereby regularly forming a plurality of openings through which the support is exposed in the sample carrier, placing the sample carrier on the sample stage, scanning the shading estimation device with the laser beam emitted from the at least one laser stimulating ray source via the openings to stimulate the support at the plurality of openings, photoelectrically detecting fluorescence emission released from the support via the plurality of openings by the light detector to produce analog data, digitizing the analog data, to store the shading correction data of the shading estimation device in the non-volatile memory or the memory of the data processing apparatus, and to correct digital data of the at least one sample based on the shading correction data stored in the non-volatile memory or the memory of the data processing apparatus, and, therefore, it is possible to produce the correction data by scanning the shading estimation device obtained by providing the mask of metal on the support capable of being processed while retaining optical flatness, thereby forming the plurality of openings through which the support is exposed with the laser beam to store them in the non-volatile memory or the memory of the data processing apparatus and produce digital data of the at least one sample in which shading has been corrected using the shading correction data in the non-volatile memory or the memory of the data processing apparatus.
In a further preferred aspect of the present invention, digital data of the shading estimation device are produced by integrating signal intensity of digital signals obtained by the photoelectrically detecting fluorescence emission released from the support through every opening.
In a further preferred aspect of the present invention, each of the plurality of openings is constituted as a slit.
In a further preferred aspect of the present invention, each of the plurality of openings is constituted as a pinhole.
In a further preferred aspect of the present invention, the digital data of the shading estimation device are produced by scanning the shading estimation device with the laser beam via the plurality of openings after a focus of the confocal optical system is adjusted to the opening located at the reference position among the plurality of openings regularly formed in the shading estimation device, photoelectrically detecting fluorescence emission released from the support to produce analog data and digitizing the analog data.
According to this preferred aspect of the present invention, since the focus of the confocal optical system is adjusted to the opening located at the reference position in an optimum manner, the signal intensity of digital data produced by stimulating the support at the openings other than the opening located at the reference position and photoelectrically detecting fluorescence emission released from the support is lower than the signal intensity of digital data produced by stimulating the support at the opening located at the reference position and photoelectrically detecting fluorescence emission released from the support. Therefore, the shading of the digital data of the at least one sample can be effectively corrected by producing shading correction data which can correct the digital data so that the signal intensity of digital data produced by stimulating the support at the openings other than the opening located at the reference position and photoelectrically detecting fluorescence emission released from the support becomes equal to the signal intensity of digital data produced by stimulating the support at the opening located at the reference position and photoelectrically detecting fluorescence emission released from the support, correcting the digital data of the at least one sample using the thus produced shading correction data.
In a further preferred aspect of the present invention, the shading correction data are produced for each wavelength of the laser beam and stored in the non-volatile memory or the memory of the data processing apparatus.
Although the focus position of the confocal optical system depends upon the wavelengths of laser beams and the shading generated in digital data changes in accordance with the wavelengths of laser beams, according to this preferred aspect of the present invention, it is possible to correct the shading of digital data of the at least one sample when the laser beam having a wavelength capable of most efficiently stimulating a labeling substance such as a fluorescent substance is selected to stimulate at least one sample and fluorescence emission released from the sample is photoelectrically detected, because the shading correction data are produced for each wavelength of the laser beam and stored in the non-volatile memory or the memory of the data processing apparatus.
In a further preferred aspect of the present invention, the support is formed of a material selected from a group consisting of IV group elements, II-VI group compounds, III-V group compounds and complexes thereof.
In a further preferred aspect of the present invention, the support is formed by a color glass filter formed by doping glass containing a material selected from a group consisting of silica sand, soda ash and limestone as a principal component with a solid solution of CdSxe2x80x94CdSe.
According to this preferred aspect of the present invention, since the color glass filter formed by doping glass containing a material selected from a group consisting of silica sand, soda ash and limestone as a principal component with a solid solution of CdSxe2x80x94CdSe can be processed so as to retain optical flatness and has a property to release fluorescence emission upon being irradiated with a laser beam and a plurality of openings through which the color glass filter is exposed can be formed by providing a mask of metal on the color glass filter, it is possible to produce digital data of the at least one sample in which the shading has been accurately corrected by scanning the color glass filter via the plurality of regularly formed openings, photoelectrically detecting fluorescence emission released from the color glass filter at the plurality of openings to produce shading correction data, storing them in the non-volatile memory or the memory of the data processing apparatus and correcting the digital data of the at least one sample using the shading correction data.
In a further preferred aspect of the present invention, the support is formed by a color glass filter formed by doping glass containing a material selected from a group consisting of silica sand, soda ash and limestone as a principal component with a solid solution of ZnSxe2x80x94CdS.
According to this preferred aspect of the present invention, since the color glass filter formed by doping glass containing a material selected from a group consisting of silica sand, soda ash and limestone as a principal component with a solid solution of ZnSxe2x80x94CdS can be processed so as to retain optical flatness and has a property to release fluorescence emission upon being irradiated with a laser beam and a plurality of openings through which the color glass filter is exposed can be formed by providing a mask of metal on the color glass filter, it is possible to produce digital data of the at least one sample in which the shading has been accurately corrected by scanning the color glass filter via the plurality of regularly formed openings, photoelectrically detecting fluorescence emission released from the color glass filter at the plurality of openings to produce shading correction data, storing them in the non-volatile memory or the memory of the data processing apparatus and correcting the digital data of the at least one sample using the shading correction data stored in the non-volatile memory or the memory of the data processing apparatus.
In a further preferred aspect of the present invention, the support is formed by a laminate of an InGaAsP layer and a GaAs layer and the metal layer is formed on the InGaAsP layer.
According to this preferred aspect of the present invention, since the laminate of an InGaAsP layer and a GaAs layer can be processed so as to retain optical flatness and has a property to release fluorescence emission upon being irradiated with a laser beam and a plurality of openings through which the color glass filter is exposed can be formed by providing a mask of metal on the color glass filter, it is possible to produce digital data of the at least one sample in which the shading has been accurately corrected by scanning the color glass filter via the plurality of regularly formed openings, photoelectrically detecting fluorescence emission released from the color glass filter at the plurality of openings to produce shading correction data, storing them in the non-volatile memory or the memory of the data processing apparatus and correcting the digital data of the at least one sample using the shading correction data stored in the non-volatile memory or the memory of the data processing apparatus.
In a further preferred aspect of the present invention, the mask of metal is formed by a method selected from a group consisting of sputtering, chemical vapor deposition and evaporation.
In a further preferred aspect of the present invention, the mask of metal is formed of a material selected from a group consisting of chromium, aluminum, gold, nickel-chromium alloy and titanium-nickel-chromium alloy.
The above and other objects of the present invention can be also accomplished by a method for producing focus position data of a confocal optical system of a scanner having a confocal optical system comprising steps of setting at least one distance measuring device in a sample carrier for carrying at least one sample, placing the sample carrier on the sample stage, measuring a distance between an objective lens incorporated in the confocal optical system and a reference position on a surface of the at least one distance measuring device set in the sample carrier and a distance between the objective lens and at least one measurement position on a surface of the at least one distance measuring device different from the reference position, thereby producing position data, storing them in a non-volatile memory, setting a focus position determination device including a luminescent material having a property to release fluorescence emission or photoluminescence emission upon being irradiated with the laser beam in the sample carrier so that the luminescent material is located at the reference position, scanning the focus position determination device with the laser beam to stimulate the luminescent material located at the reference position, photoelectrically detecting fluorescence emission or photoluminescence emission released from the luminescent material, changing the position of the objective lens of the confocal optical system with a predetermined pitch, determining a focus position of the confocal optical system, thereby producing the position data, and storing them in the non-volatile memory.
According to this preferred aspect of the present invention, the method for producing focus position data of a confocal optical system of a scanner having a confocal optical system comprises the steps of setting at least one distance measuring device in the sample carrier for carrying at least one sample, placing the sample carrier on the sample stage, measuring a distance between the objective lens incorporated in the confocal optical system and a reference position on a surface of the at least one distance measuring device set in the sample carrier and a distance between the objective lens and at least one measurement position on a surface of the at least one distance measuring device different from the reference position thereby producing position data, storing them in a non-volatile memory, setting focus position determination device including a luminescent material having a property to release fluorescence emission or photoluminescence emission upon being irradiated with the laser beam in the sample carrier so that the luminescent material is located at the reference position, scanning the focus position determination device with the laser beam to stimulate the luminescent material located at the reference position, photoelectrically detecting fluorescence emission or photoluminescence emission released from the luminescent material, changing the position of the objective lens of the confocal optical system with a predetermined pitch, determining a focus position of the confocal optical system, thereby producing the position data, and storing them in the non-volatile memory, and, therefore, the objective lens of the confocal optical system can be moved to adjust the position of the objective lens based on the focus position of the confocal is optical system obtained by correcting the focus position of the confocal optical system read from the non-volatile memory in accordance with the position data read from the non-volatile memory, so that the focus of the confocal optical system can be adjusted in a desired manner without an auto-focusing system provided with special devices such as a reflected light detecting optical system, a sensor, a detection circuit and the like.
In a preferred aspect of the present invention, the position of the objective lens of the confocal optical system at which an integrated value of signal intensity of fluorescence emission or photoluminescence emission detected by the light detector becomes maximum is determined as the focus position of the confocal optical.
In a further preferred aspect of the present invention, the method for producing focus position data of a confocal optical system of a scanner having a confocal optical system comprises steps of measuring the distance between the objective lens incorporated in the confocal optical system and the reference position on the surface of the at least one distance measuring device set in the sample carrier and the distance between the objective lens and the at least one measurement position on the surface of the at least one distance measuring device different from the reference position and calculating a displacement of the at least one measurement position on the surface of the at least one distance measuring device set in the sample carrier different from the reference position with respect to the reference position on the surface of the at least one distance measuring device, thereby producing the position data.
In a further preferred aspect of the present invention, the method for producing focus position data of a confocal optical system of a scanner having a confocal optical system comprises steps of measuring the distance between the objective lens incorporated in the confocal optical system and the reference position on the surface of the at least one distance measuring device set in the sample carrier and the distance between the objective lens and two or more measurement positions different from the reference position on the surface of the at least one distance measuring device, calculating displacements of the two or more measurement positions different from the reference position on the surface of the at least one distance measuring device set in the sample carrier with respect to the reference position on the surface of the at least one distance measuring device, and averaging the displacements of the two or more measurement positions different from the reference position on the surface of the at least one distance measuring device with respect to the reference position, thereby producing the position data.
According to this preferred aspect of the present invention, the focus of the confocal system can be more accurately adjusted because the method for producing focus position data of a confocal optical system of a scanner having a confocal optical system comprises steps of measuring the distance between the objective lens incorporated in the confocal optical system and the reference position on the surface of the at least one distance measuring device set in the sample carrier and the distance between the objective lens and two or more measurement positions different from the reference position on the surface of the at least one distance measuring device, calculating displacements of the two or more measurement positions different from the reference position on the surface of the at least one distance measuring device set in the sample carrier with respect to the reference position on the surface of the at least one distance measuring device, and averaging the displacements of the two or more measurement positions different from the reference position on the surface of the at least one distance measuring device with respect to the reference position, thereby producing the position data.
In a further preferred aspect of the present invention, the method for producing focus position data of a confocal optical system of a scanner having a confocal optical system comprises steps of measuring the distance between the objective lens incorporated in the confocal optical system and the reference position on the surface of the at least one distance measuring device set in the sample carrier and the distance between the objective lens and two or more measurement positions different from the reference position on the surface of the at least one distance measuring device at two or more different temperatures from each other, calculating displacements of the two or more measurement positions different from the reference position on the surface of the at least one distance measuring device set in the sample carrier with respect to the reference position on the surface of the at least one distance measuring device, and storing temperature coefficients of the displacements of the two or more measurement positions different from the reference position on the surface of the at least one distance measuring device with respect to the reference position and an average temperature in the scanner when the focus position data were produced in the non-volatile memory.
According to this preferred aspect of the present invention, the method for producing focus position data of a confocal optical system of a scanner having a confocal optical system comprises steps of measuring the distance between the objective lens incorporated in the confocal optical system and the reference position on the surface of the at least one distance measuring device set in the sample carrier and the distance between the objective lens and two or more measurement positions different from the reference position on the surface of the at least one distance measuring device at two or more different temperatures from each other, calculating displacements of the two or more measurement positions different from the reference position on the surface of the at least one distance measuring device set in the sample carrier with respect to the reference position on the surface of the at least one distance measuring device, and storing temperature coefficients of the displacements of the two or more measurement positions different from the reference position on the surface of the at least one distance measuring device with respect to the reference position and an average temperature in the scanner when the focus position data were produced in the non-volatile memory, and, therefore, it is possible to adjust the focus of the confocal optical system in a desired manner even when temperature in the scanner changes by correcting the focus position data of the confocal optical system read from the non-volatile memory according to difference in temperature between the temperature in the scanner and the average temperature in the scanner when the focus position data of the confocal optical system were produced and in accordance with the temperature coefficients of the displacements of the two or more measurement positions different from the reference position on the surface of the at least one distance measuring device with respect to the reference position stored in the non-volatile memory, and moving the objective lens of the confocal optical system based on the thus corrected focus position data of the confocal optical system.
In a further preferred aspect of the present invention, the sample carrier is constituted so as to carry two or more distance measuring devices and the method for producing focus position data of a confocal optical system of a scanner comprises steps of placing the sample carrier carrying two or more samples on the sample stage, measuring distances between the objective lens incorporated in the confocal optical system and three or more measurement positions different from each other on each of the two or more distance measuring devices, determining one of the measurement positions on one of the two or more distance measuring devices as the reference position, calculating displacements of the measurement positions on each of the two or more distance measuring devices with respect to the reference position, averaging the displacements of the measurement positions on each of the two or more distance measuring devices to produce the position data, and storing them in the non-volatile memory.
According to this preferred aspect of the present invention, the sample carrier is constituted so as to carry two or more distance measuring devices and the method for producing focus position data of a confocal optical system of a scanner comprises steps of placing the sample carrier carrying two or more samples on the sample stage, measuring distances between the objective lens incorporated in the confocal optical system and three or more measurement positions different from each other on each of the two or more distance measuring devices, determining one of the measurement positions on one of the two or more distance measuring devices as the reference position, calculating displacements of the measurement positions on each of the two or more distance measuring devices with respect to the reference position, averaging the displacements of the measurement positions on each of the two or more distance measuring devices to produce the position data, and storing them in the non-volatile memory, and, therefore, it is possible to accurately adjust the focus of the confocal system in a desired manner for each of the plurality of samples set in the sample carrier by correcting the focus position data of the confocal optical system read from the non-volatile memory based on the position data read from the non-volatile memory for each of samples set at positions corresponding to positions of the two or more distance measuring devices set in the sample carrier and moving the objective lens of the confocal optical system in accordance with the thus corrected focus position data of the confocal optical system, thereby adjusting the position of the objective lens.
In a further preferred aspect of the present invention, the method for producing focus position data of a confocal optical system of a scanner having a confocal optical system comprises steps of measuring distances between the objective lens incorporated in the confocal optical system and the three or more measurement positions on each of the two or more distance measuring devices with respect to the reference position determined by one of the measurement positions on one of the two or more distance measuring devices at two or more temperatures different from each other, calculating displacements of the measurement positions on each of the two or more distance measuring devices with respect to the reference position to calculate the temperature coefficients of displacements of the measurement positions with respect to the reference position, and storing the thus calculated temperature coefficients and an average temperature in the scanner when the focus position data were produced in the non-volatile memory.
According to this preferred aspect of the present invention, the method for producing focus position data of a confocal optical system of a scanner having a confocal optical system comprises steps of measuring distances between the objective lens incorporated in the confocal optical system and the three or more measurement positions on each of the two or more distance measuring devices with respect to the reference position determined by one of the measurement positions on one of the two or more distance measuring devices at two or more temperatures different from each other, calculating displacements of the measurement positions on each of the two or more distance measuring devices with respect to the reference position to calculate the temperature coefficients of displacements of the measurement positions with respect to the reference position, and storing the thus calculated temperature coefficients and an average temperature in the scanner when the focus position data were produced in the non-volatile memory, and, therefore, it is possible to accurately adjust the focus of the confocal system for each of the plurality of samples set in the sample carrier even when the temperature in the scanner changes by correcting the focus position data of the confocal optical system read from the non-volatile memory for each of the two or more distance measuring devices according to difference in temperature between the temperature in the scanner and the average temperature in the scanner when the focus position data of the confocal optical system were produced and in accordance with the temperature coefficients of the two or more distance measuring devices read from the non-volatile memory and moving the objective lens of the confocal optical system in accordance with the thus corrected focus position data of the confocal optical system, thereby adjusting the position of the objective lens.
In a further preferred aspect of the present invention, the method for producing focus position data of a confocal optical system of a scanner having a confocal optical system comprises steps of measuring distances between the objective lens incorporated in the confocal optical system and at least two measurement positions different from each other in each main scanning line on each of the two or more distance measuring devices set in the sample carrier, determining one of the measurement positions on one of the two or more distance measuring devices as the reference position, calculating displacements of the measurement positions on each of the two or more distance measuring devices with respect to the reference position, averaging the displacements of the measurement positions on each of the two or more distance measuring devices to produce the position data, and storing the thus produced position data in the non-volatile memory.
In a further preferred aspect of the present invention, the method for producing focus position data of a confocal optical system of a scanner having a confocal optical system comprises steps of measuring distances between the objective lens incorporated in the confocal optical system and the at least two measurement positions in each main scanning line on each of the two or more distance measuring devices with respect to the reference position at two or more temperatures different from each other, determining one of the measurement positions on one of the two or more distance measuring devices as the reference position, calculating displacements of the measurement positions on each of the two or more distance measuring devices with respect to the reference position to produce the temperature coefficients of displacements of the measurement positions with respect to the reference position, and storing an average value of the temperature coefficients of displacements of the measurement positions with respect to the reference position as a temperature coefficient and an average temperature in the scanner when the focus position data were produced in the non-volatile memory.
According to this preferred aspect of the present invention, the method for producing focus position data of a confocal optical system of a scanner having a confocal optical system comprises steps of measuring distances between the objective lens incorporated in the confocal optical system and the at least two measurement positions in each main scanning line on each of the two or more distance measuring devices with respect to the reference position at two or more temperatures different from each other, determining one of the measurement positions on one of the two or more distance measuring devices as the reference position, calculating displacements of the measurement positions on each of the two or more distance measuring devices with respect to the reference position to produce the temperature coefficients of displacements of the measurement positions with respect to the reference position, and storing an average value of the temperature coefficients of displacements of the measurement positions with respect to the reference position as a temperature coefficient and an average temperature in the scanner when the focus position data were produced in the non-volatile memory, and, therefore, it is possible to more accurately adjust the focus of the confocal system for each of the plurality of samples set in the sample carrier even when the temperature in the scanner changes by correcting the focus position data of the confocal optical system read from the non-volatile memory for each of the two or more distance measuring devices according to difference in temperature between the temperature in the scanner and the average temperature in the scanner when the focus position data of the confocal optical system were produced and in accordance with the temperature coefficients of the two or more distance measuring devices read from the non-volatile memory and moving the objective lens of the confocal optical system in accordance with the thus corrected focus position data of the confocal optical system, thereby adjusting the position of the objective lens.
In a further preferred aspect of the present invention, the method for producing focus position data of a confocal optical system of a scanner having a confocal optical system comprises steps of measuring distances between the objective lens incorporated in the confocal optical system and three measurement positions different from each other in each main scanning line on each of the two or more distance measuring devices set in the sample carrier, determining one of the measurement positions on one of the two or more distance measuring devices as the reference position, calculating displacements of the measurement positions on each of the two or more distance measuring devices with respect to the reference position, averaging the displacements of the measurement positions on each of the two or more distance measuring devices to produce the position data, and storing the thus produced position data in the non-volatile memory.
In a further preferred aspect of the present invention, the method for producing focus position data of a confocal optical system of a scanner having a confocal optical system comprises steps of measuring distances between the objective lens incorporated in the confocal optical system and the three measurement positions in each main scanning line on each of the two or more distance measuring devices at two or more temperatures different from each other, determining one of the measurement positions on one of the two or more distance measuring devices as the reference position, calculating displacements of the measurement positions on each of the two or more distance measuring devices with respect to the reference position to produce the temperature coefficients of displacements of the measurement positions with respect to the reference position, and storing an average value of the temperature coefficients of displacements of the measurement positions with respect to the reference position as a temperature coefficient and an average temperature in the scanner when the focus position data were produced in the non-volatile memory.
In a further preferred aspect of the present invention, the method for producing focus position data of a confocal optical system of a scanner having a confocal optical system comprises steps of averaging displacements of the measurement positions with respect to the reference position for every predetermined number of the main scanning lines on each of the two or more distance measuring devices to produce the position data and storing the thus produced position data.
According to this preferred aspect of the present invention, since the method for producing focus position data of a confocal optical system of a scanner having a confocal optical system comprises steps of averaging displacements of the measurement positions with respect to the reference position for every predetermined number of the main scanning lines on each of the two or more distance measuring devices to produce the position data and storing the thus produced position data, the position of the objective lens can be more precisely adjusted and it is therefore possible to more accurately adjust the focus of the confocal system for each of the plurality of samples set in the sample carrier.
In a further preferred aspect of the present invention, the method for producing focus position data of a confocal optical system of a scanner having a confocal optical system comprises steps of measuring distances between the objective lens incorporated in the confocal optical system and three measurement positions in each main scanning line for each predetermined number of main scanning lines on each of the two or more distance measuring devices at two or more temperatures different from each other, determining one of the measurement positions on one of the two or more distance measuring devices as the reference position, calculating displacements of the measurement positions with respect to the reference position for every predetermined number of the main scanning lines on each of the two or more distance measuring devices to produce the temperature coefficients of displacements of the measurement positions with respect to the reference position, and storing an average value of the temperature coefficients of displacements of the measurement positions with respect to the reference position every predetermined number of the main scanning lines on each of the two or more distance measuring devices as a temperature coefficient and an average temperature in the scanner when the focus position data were produced in the non-volatile memory.
According to this preferred aspect of the present invention, the method for producing focus position data of a confocal optical system of a scanner having a confocal optical system comprises steps of measuring distances between the objective lens incorporated in the confocal optical system and three measurement positions in each main scanning line for each predetermined number of main scanning lines on each of the two or more distance measuring devices at two or more temperatures different from each other, determining one of the measurement positions on one of the two or more distance measuring devices as the reference position, calculating displacements of the measurement positions with respect to the reference position for every predetermined number of the main scanning lines on each of the two or more distance measuring devices to produce the temperature coefficients of displacements of the measurement positions with respect to the reference position, and storing an average value of the temperature coefficients of displacements of the measurement positions with respect to the reference position every predetermined number of the main scanning lines on each of the two or more distance measuring devices as a temperature coefficient and an average temperature in the scanner when the focus position data were produced in the non-volatile memory, and, therefore, the position of the objective lens can be more precisely adjusted even when the temperature in the scanner changes by correcting the focus position data of the confocal optical system read from the non-volatile memory for each number of main scanning lines corresponding to the predetermined number of main scanning lines of the samples set at positions in the sample carrier corresponding to positions where the two or more distance measuring devices were set according to difference in temperature between the temperature in the scanner and the average value of temperatures in the scanner when the focus position data of the confocal optical system were produced and in accordance with the temperature coefficients of the predetermined number of main scanning lines on each of the two or more distance measuring devices read from the non-volatile memory, and moving the objective lens of the confocal optical system in accordance with the thus corrected focus position data of the confocal optical system, thereby adjusting the position of the objective lens, and it is therefore possible to more accurately adjust the focus of the confocal system for each of the plurality of samples set in the sample carrier.
In a further preferred aspect of the present invention, the focus position data of the confocal optical system are produced for each wavelength of a laser beam and stored in the non-volatile memory.
Although the focus position of the confocal optical system depends upon the wavelength of the laser beam, i.e., the stimulating ray, according to this preferred aspect of the present invention, the focus position data of the confocal optical system are produced for each wavelength of a laser beam and stored in the non-volatile memory, and, therefore, when the laser beam having a wavelength capable of most efficiently stimulating a labeling substance, a fluorescent dye, is selected to stimulate a sample(s) and fluorescence emission released from the sample is photoelectrically detected, the focus position of the confocal optical system can be adjusted in a desired manner by reading the focus position data of the confocal optical system corresponding to the wavelength of a laser beam emitted from the laser stimulating ray source to be used for scanning the at least one sample among two or more laser stimulating ray sources from the non-volatile memory, and moving the objective lens of the confocal optical system based on the thus read focus position data of the confocal optical system, thereby adjusting the position of the objective lens.
In a further preferred aspect of the present invention, the distance measuring device is provided with a metal layer on the surface thereof and the position data are produced using an electrostatic capacitance displacement meter.
According to this preferred aspect of the present invention, since the distance measuring device is provided with a metal layer on the surface thereof and the position data are produced using an electrostatic capacitance displacement meter, it is possible to produce the position data on the sub-micron order to enable accurate adjustment of the focus position of the confocal optical system.
In a further preferred aspect of the present invention, the position data are optically produced.
According to this preferred aspect of the present invention, in the case where the position data are produced using an auto-focusing system, a laser displacement meter or the like and the thus produced position data are stored in the non-volatile memory, the scanner itself does not require auto-focusing capability, and, therefore, it is possible to adjust the focus position of the confocal optical system in a desired manner without need for an auto-focusing system including special devices such as a reflected light detection system, a sensor, a detection circuit and the like.
In a further preferred aspect of the present invention, the metal layer of the distance measuring device is formed by a method selected from a group consisting of sputtering, chemical vapor deposition and evaporation.
In a further preferred aspect of the present invention, the metal layer of the distance measuring device is formed by sputtering.
In a further preferred aspect of the present invention, the metal layer of the distance measuring device is formed of a material selected from a group consisting of chromium, aluminum, gold, nickel-chromium alloy and titanium-nickel-chromium alloy.
In a further preferred aspect of the present invention, the metal layer of the distance measuring device is formed of chromium.
In a further preferred aspect of the present invention, the focus position determination device is constituted as a slide glass plate including a spot containing at least one kind of fluorescent dye formed on the surface thereof.
In a further preferred aspect of the present invention, the spot contains two or more kinds of fluorescent dyes capable of efficiently being stimulated by laser beams having different wavelengths from each other.
Although the focus position of the confocal optical system depends upon the wavelength of the laser beam, i.e., the stimulating ray, according to this preferred aspect of the present invention, since the spot contains two or more kinds of fluorescent dyes capable of efficiently being stimulated by laser beams having different wavelengths from each other, it is possible to produce the focus position data of the confocal optical system for each wavelength of the laser beam and store them in the non-volatile memory and, therefore, it is possible to adjust the focus position of the confocal optical system in a desired manner when the laser beam having a wavelength capable of most efficiently stimulating a labeling substance, a fluorescent dye, is selected to stimulate a sample(s) and fluorescence emission released from the sample is photoelectrically detected.
In a further preferred aspect of the present invention, the spot contains a fluorescent dye capable of being efficiently stimulated by a laser beam having a wavelength between 470 nm and 490 nm, a fluorescent dye capable of being efficiently stimulated by a laser beam having a wavelength between 530 nm and 540 nm and a fluorescent dye capable of being efficiently stimulated by a laser beam having a wavelength between 630 nm and 650 nm.
Although the focus position of the confocal optical system depends upon the wavelength of the laser beam i.e., the stimulating ray, according to this preferred aspect of the present invention, the focus position of the confocal optical system can be adjusted in a desired manner when the laser beam having a wavelength capable of most efficiently stimulating a typical fluorescent dye is selected to stimulate a sample(s) and fluorescence emission released from the sample is photoelectrically detected, because the focus position data of the confocal optical system can be produced for a laser beam having a wavelength between 470 nm and 490 nm, a laser beam having a wavelength between 530 nm and 540 nm and having a wavelength between 630 nm and 650 nm using the spot containing a fluorescent dye capable of being efficiently stimulated by a laser beam having a wavelength between 470 nm and 490 nm, a fluorescent dye capable of being efficiently stimulated by a laser beam having a wavelength between 530 nm and 540 nm and a fluorescent dye capable of being efficiently stimulated by a laser beam having a wavelength between 630 nm and 650 nm and stored in the non-volatile memory.
In a further preferred aspect of the present invention, the focus position determination device is formed by providing the metal layer on a support formed of an inorganic material which can release fluorescence emission or photoluminescence emission and is not degraded by the irradiation with the laser beam so as to form an opening through which the support is exposed.
Although a fluorescent dye is sometimes degraded with the lapse of time when being irradiated with a laser beam, whereby the amount of released fluorescence emission is lowered and the focus position of the confocal optical system cannot be determined in a desired manner, according to this preferred aspect of the present invention, since the focus position determination device is formed by providing the metal layer on a support formed of an inorganic material which can release fluorescence emission or photoluminescence emission and is not degraded by the irradiation with the laser beam so as to form an opening through which the support is exposed, it is possible to accurately determine the focus position of the confocal optical system in a desired manner.
In a further preferred aspect of the present invention, the support is formed of a material selected from a group consisting of IV group elements, II-VI group compounds, III-V group compounds and complexes thereof.
In a further preferred aspect of the present invention, the support is formed by a color glass filter formed by doping glass containing a material selected from a group consisting of silica sand, soda ash and limestone as a principal component with a solid solution of CdSxe2x80x94CdSe.
Although a fluorescent dye is sometimes degraded with the lapse of time when being irradiated with a laser beam, whereby the amount of released fluorescence emission is lowered and the focus position of the confocal optical system cannot be determined in a desired manner, according to this preferred aspect of the present invention, since the support is formed by a color glass filter formed by doping glass containing a material selected from a group consisting of silica sand, soda ash and limestone as a principal component with a solid solution of CdSxe2x80x94CdSe and is not degraded by the irradiation with the laser beam, it is possible to accurately determine the focus position of the confocal optical system in a desired manner.
In a further preferred aspect of the present invention, the support is formed by a color glass filter formed by doping glass containing a material selected from a group consisting of silica sand, soda ash and limestone as a principal component with a solid solution of ZnSxe2x80x94CdS.
Although a fluorescent dye is sometimes degraded with the lapse of time when being irradiated with a laser beam, whereby the amount of released fluorescence emission is lowered and the focus position of the confocal optical system cannot be determined in a desired manner, according to this preferred aspect of the present invention, since the support is formed by a color glass filter formed by doping glass containing a material selected from a group consisting of silica sand, soda ash and limestone as a principal component with a solid solution of ZnSxe2x80x94CdS and is not degraded by the irradiation with the laser beam, it is possible to accurately determine the focus position of the confocal optical system in a desired manner.
In a further preferred aspect of the present invention, the support is formed by a laminate of an InGaAsP layer and a GaAs layer and the metal layer is formed on the InGaAsP layer.
Although a fluorescent dye is sometimes degraded with the lapse of time when being irradiated with a laser beam, whereby the amount of released fluorescence emission is lowered and the focus position of the confocal optical system cannot be determined in a desired manner, according to this preferred aspect of the present invention, since the support is formed by a laminate of an InGaAsP layer and a GaAs layer which are not degraded by the irradiation with the laser beam and the metal layer is formed on the InGaAsP layer, it is possible to accurately determine the focus position of the confocal optical system in a desired manner.
In a further preferred aspect of the present invention, the metal layer is formed by a method selected from a group consisting of sputtering, chemical vapor deposition and evaporation.
In a further preferred aspect of the present invention, the metal layer is formed by sputtering.
In a further preferred aspect of the present invention, the metal layer is formed of a material selected from a group consisting of chromium, aluminum, gold, nickel-chromium alloy and titanium-nickel-chromium alloy.
In a further preferred aspect of the present invention, the metal layer is formed of chromium.
In a further preferred aspect of the present invention, the objective lens of the confocal optical system is adjusted by a stepping motor.
In a further preferred aspect of the present invention, the focus position data are produced in the form of drive pulses to be applied to the stepping motor and stored in the non-volatile memory, the number of drive pulses stored in the non-volatile memory is corrected to be applied to the stepping motor and the objective lens of the confocal optical system is moved to adjust the position of the objective lens.
In a further preferred aspect of the present invention, the method for producing focus position data of a confocal optical system of a scanner having a confocal optical system further comprises steps of plotting integrated values of signal intensity of fluorescence emission or photoluminescence emission, fitting the plotted values with the nth order function to produce coefficients of the nth order function and storing the coefficients of the nth order function in the non-volatile memory as well as displacements of measurement positions on the at least one distance measuring device with respect to the reference position.
According to this preferred aspect of the present invention, the method for producing focus position data of a confocal optical system of a scanner having a confocal optical system further comprises steps of plotting integrated values of signal intensity of fluorescence emission or photoluminescence emission, fitting the plotted values with the nth order function to produce coefficients of the nth order function and storing the coefficients of the nth order function in the non-volatile memory as well as displacements of measurement positions on the at least one distance measuring device with respect to the reference position, and, therefore, it is possible to produce the shading correction data as well as the focus position data of the confocal optical system and produce the digital data of the at least one sample in which the shading has been corrected by producing shading correction data for correcting shading of digital data of the at least one sample based on the coefficients of the nth order function and the displacements of measurement positions on the at least one distance measuring device with respect to the reference position read from the non-volatile memory and correcting the digital data of the at least one sample based on the shading correction data.
In a further preferred aspect of the present invention, the method for producing focus position data of a confocal optical system of a scanner having a confocal optical system further comprises steps of plotting integrated values of signal intensity of detected fluorescence emission or photoluminescence emission, fitting the plotted values with the nth order function to produce coefficients of the nth order function, producing shading correction data for correcting shading of digital data of the at least one sample based on the coefficients of the nth order function and the displacements of measurement positions on the at least one distance measuring device with respect to the reference position and storing the thus produced shading correction data in the non-volatile memory.
According to this preferred aspect of the present invention, it is possible to produce the shading correction data as well as the focus position data of the confocal optical system and produce the digital data of the sample in which the shading has been corrected, because the method for producing focus position data of a confocal optical system of a scanner having a confocal optical system further comprises steps of plotting integrated values of signal intensity of detected fluorescence emission or photoluminescence emission, fitting the plotted values with the nth order function to produce coefficients of the nth order function, producing shading correction data for correcting shading of digital data of the at least one sample based on the coefficients of the nth order function and the displacements of measurement positions on the at least one distance measuring device with respect to the reference position and storing the thus produced shading correction data in the non-volatile memory.
In a further preferred aspect of the present invention, the method for producing focus position data of a confocal optical system of a scanner having a confocal optical system further comprises steps of setting a shading estimation device in which a mask of metal is formed on a support having a property to release fluorescence emission upon being irradiated with a laser beam and capable of being processed while retaining optical flatness, thereby regularly forming a plurality of openings through which the support is exposed in the sample carrier, placing the sample carrier on the sample stage, scanning the shading estimation device with the laser beam via the openings to stimulate the support at the plurality of openings, photoelectrically detecting fluorescence emission released from the support via the plurality of openings to produce analog data, digitizing the analog data, producing digital data of the shading estimation device based on the thus produced digital data, producing shading correction data based on the digital data of the shading estimation device, and storing the thus produced shading correction data in the non-volatile memory or the memory of the scanner.
According to this preferred aspect of the present invention, it is possible to correct digital data of at least one sample by reading shading correction data stored in the non-volatile memory or the memory of the scanner because the method for producing focus position data of a confocal optical system of a scanner having a confocal optical system further comprises steps of setting a shading estimation device in which a mask of metal is formed on a support having a property to release fluorescence emission upon being irradiated with a laser beam and capable of being processed while retaining optical flatness, thereby regularly forming a plurality of openings through which the support is exposed in the sample carrier, placing the sample carrier on the sample stage, scanning the shading estimation device with the laser beam via the openings to stimulate the support at the plurality of openings, photoelectrically detecting fluorescence emission released from the support via the plurality of openings to produce analog data, digitizing the analog data, producing digital data of the shading estimation device based on the thus produced digital data, producing shading correction data based on the digital data of the shading estimation device, and storing the thus produced shading correction data in the non-volatile memory or the memory of the scanner.
In a further preferred aspect of the present invention, digital data of the shading estimation device are produced by integrating signal intensity of digital signals obtained by the photoelectrically detecting fluorescence emission released from the support through every opening.
In a further preferred aspect of the present invention, each of the plurality of openings is constituted as a slit.
In a further preferred aspect of the present invention, each of the plurality of openings is constituted as a pinhole.
In a further preferred aspect of the present invention, the method for producing focus position data of a confocal optical system of a scanner having a confocal optical system further comprises steps of adjusting a focus of the confocal optical system to the opening located at the reference position among the plurality of openings regularly formed in the shading estimation device, scanning the shading estimation device with the laser beam via the plurality of openings, stimulating the support via the plurality of openings, photoelectrically detecting fluorescence emission released from the support to produce analog data, and digitizing the analog data to produce digital data of the shading estimation device.
According to this preferred aspect of the present invention, since the focus of the confocal optical system is adjusted to the opening located at the reference position in an optimum manner, the signal intensity of digital data produced by stimulating the support at openings other than the opening located at the reference position and photoelectrically detecting fluorescence emission released from the support is lower than the signal intensity of digital data produced by stimulating the support at the opening located at the reference position and photoelectrically detecting fluorescence emission released from the support. Therefore, the shading of the digital data of the at least one sample can be effectively corrected by producing shading correction data which can correct the digital data so that the signal intensity of digital data produced by stimulating the support at openings other than the opening located at the reference position and photoelectrically detecting fluorescence emission released from the support becomes equal to the signal intensity of digital data produced by stimulating the support at the opening located at the reference position and photoelectrically detecting fluorescence emission released from the support, correcting the digital data of the at least one sample using the thus produced shading correction data.
In a further preferred aspect of the present invention, the shading correction data are produced for each wavelength of the laser beam and stored in the non-volatile memory or the memory of the scanner.
Although the focus position of the confocal optical system depends upon the wavelengths of laser beams and the shading generated in digital data changes in accordance with the wavelengths of laser beams, according to this preferred aspect of the present invention, it is possible to correct the shading of digital data of the at least one sample when the laser beam having a wavelength capable of most efficiently stimulating a labeling substance such as a fluorescent substance is selected to stimulate at least one sample and fluorescence emission released from the sample is photoelectrically detected, because the shading correction data are produced for each wavelength of the laser beam and stored in the non-volatile memory or the memory of the scanner.
In a further preferred aspect of the present invention, the support is formed of a material selected from a group consisting of IV group elements, II-VI group compounds, III-V group compounds and the complexes thereof.
In a further preferred aspect of the present invention, the support is formed by a color glass filter formed by doping glass containing a material selected from a group consisting of silica sand, soda ash and limestone as a principal component with a solid solution of CdSxe2x80x94CdSe.
According to this preferred aspect of the present invention, since the color glass filter formed by doping glass containing a material selected from a group consisting of silica sand, soda ash and limestone as a principal component with a solid solution of CdSxe2x80x94CdSe can be processed so as to retain optical flatness and has a property to release fluorescence emission upon being irradiated with a laser beam and a plurality of openings through which the color glass filter is exposed can be formed by providing a mask of metal on the color glass filter, it is possible to produce digital data of the at least one sample in which the shading has been accurately corrected by scanning the color glass filter via the plurality of regularly formed openings, photoelectrically detecting fluorescence emission released from the color glass filter at the plurality of openings to produce shading correction data, storing them in the non-volatile memory or the memory of the scanner and correcting the digital data of the at least one sample using the shading correction data.
In a further preferred aspect of the present invention, the support is formed by a color glass filter formed by doping glass containing a material selected from a group consisting of silica sand, soda ash and limestone as a principal component with a solid solution of ZnSxe2x80x94CdS.
According to this preferred aspect of the present invention, since the color glass filter formed by doping glass containing a material selected from a group consisting of silica sand, soda ash and limestone as a principal component with a solid solution of ZnSxe2x80x94CdS can be processed so as to retain optical flatness and has a property to release fluorescence emission upon being irradiated with a laser beam and a plurality of openings through which the color glass filter is exposed can be formed by providing a mask of metal on the color glass filter, it is possible to produce digital data of the at least one sample in which the shading has been accurately corrected by scanning the color glass filter via the plurality of regularly formed openings, photoelectrically detecting fluorescence emission released from the color glass filter at the plurality of openings to produce shading correction data, storing them in the non-volatile memory or the memory of the scanner and correcting the digital data of the at least one sample using the shading correction data stored in the non-volatile memory or the memory of the scanner.
In a further preferred aspect of the present invention, the support is formed by a laminate of an InGaAsP layer and a GaAs layer and the metal layer is formed on the InGaAsP layer.
According to this preferred aspect of the present invention, since the laminate of an InGaAsP layer and a GaAs layer can be processed so as to retain optical flatness and has a property to release fluorescence emission upon being irradiated with a laser beam and a plurality of openings through which the color glass filter is exposed can be formed by providing a mask of metal on the color glass filter, it is possible to produce digital data of the at least one sample in which the shading has been accurately corrected by scanning the color glass filter via the plurality of regularly formed openings, photoelectrically detecting fluorescence emission released from the color glass filter at the plurality of openings to produce shading correction data, storing them in the non-volatile memory or the memory of the scanner and correcting the digital data of the at least one sample using the shading correction data stored in the non-volatile memory or the memory of the scanner.
In a further preferred aspect of the present invention, the mask of metal is formed by a method selected from a group consisting of sputtering, chemical vapor deposition and evaporation.
In a further preferred aspect of the present invention, the mask of metal is formed of a material selected from a group consisting of chromium, aluminum, gold, nickel-chromium alloy and titanium-nickel-chromium alloy.
The above and other objects of the present invention can be also accomplished by a method for producing digital data of a scanner having a confocal optical system comprising steps of reading from the non-volatile memory position data produced by setting at least one distance measuring device in the sample carrier for carrying at least one sample, placing the sample carrier on the sample stage, and measuring a distance between the objective lens incorporated in the confocal optical system and a reference position on a surface of the at least one distance measuring device set in the sample carrier and a distance between the objective lens and at least one measurement position on a surface of the at least one distance measuring device different from the reference position and stored in a non-volatile memory of a scanner, reading focus position data produced by setting a focus position determination device including a luminescent material having a property to release fluorescence emission or photoluminescence emission upon being irradiated with the laser beam in the sample carrier so that the luminescent material is located at the reference position, scanning the focus position determination device with the laser beam to stimulate the luminescent material located at the reference position, photoelectrically detecting fluorescence emission or photoluminescence emission released from the luminescent material, changing the position of the objective lens of the confocal optical system with a predetermined pitch, and determining a focus position of the confocal optical system and stored in the non-volatile memory, correcting the focus position data of the confocal optical system in accordance with the position data, and adjusting the position of the objective lens of the confocal optical system based on the thus corrected focus position data of the confocal optical system.
According to the present invention, the focus of a confocal optical system can be adjusted in a desired manner without an auto-focusing system provided with special devices such as a reflected light detecting optical system, a sensor, a detection circuit and the like, because the method for producing digital data of a scanner having a confocal optical system comprises the steps of reading position data produced by setting at least one distance measuring device in the sample carrier for carrying at least one sample, placing the sample carrier on the sample stage, and measuring a distance between the objective lens incorporated in the confocal optical system and a reference position on a surface of the at least one distance measuring device set in the sample carrier and a distance between the objective lens and at least one measurement position on a surface of the at least one distance measuring device different from the reference position thereby producing position data and stored in a non-volatile memory of a scanner from the non-volatile memory, reading focus position data produced by setting a focus position determination device including a luminescent material having a property to release fluorescence emission or photoluminescence emission upon being irradiated with the laser beam in the sample carrier so that the luminescent material is located at the reference position, scanning the focus position determination device with the laser beam to stimulate the luminescent material located at the reference position, photoelectrically detecting fluorescence emission or photoluminescence emission released from the luminescent material, changing the position of the objective lens of the confocal optical system with a predetermined pitch, and determining a focus position of the confocal optical system and stored in the non-volatile memory, correcting the focus position data of the confocal optical system in accordance with the position data, and adjusting the position of the objective lens of the confocal optical system based on the thus corrected focus position data of the confocal optical system.
In a preferred aspect of the present invention, the position data are produced by measuring the distance between the objective lens incorporated in the confocal optical system and the reference position on the surface of the at least one distance measuring device set in the sample carrier and the distance between the objective lens and the at least one measurement position on the surface of the at least one distance measuring device different from the reference position and calculating a displacement of the at least one measurement position on the surface of the at least one distance measuring device set in the sample carrier different from the reference position with respect to the reference position on the surface of the at least one distance measuring device, and are stored in the non-volatile memory.
In a further preferred aspect of the present invention, the position data are produced by measuring the distance between the objective lens incorporated in the confocal optical system and the reference position on the surface of the at least one distance measuring device set in the sample carrier and the distance between the objective lens and two or more measurement positions different from the reference position on the surface of the at least one distance measuring device, calculating displacements of the two or more measurement positions different from the reference position on the surface of the at least one distance measuring device set in the sample carrier with respect to the reference position on the surface of the at least one distance measuring device, and averaging the displacements of the two or more measurement positions different from the reference position on the surface of the at least one distance measuring device with respect to the reference position, and are stored in the non-volatile memory.
According to this preferred aspect of the present invention, the focus of the confocal system can be more accurately adjusted because the position data are produced by measuring the distance between the objective lens incorporated in the confocal optical system and the reference position on the surface of the at least one distance measuring device set in the sample carrier and the distance between the objective lens and two or more measurement positions different from the reference position on the surface of the at least one distance measuring device, calculating displacements of the two or more measurement positions different from the reference position on the surface of the at least one distance measuring device set in the sample carrier with respect to the reference position on the surface of the at least one distance measuring device, and averaging the displacements of the two or more measurement positions different from the reference position on the surface of the at least one distance measuring device with respect to the reference position, and are stored in the non-volatile memory.
In a further preferred aspect of the present invention, the method for producing digital data of a scanner having a confocal optical system further comprises steps of reading from the non-volatile memory temperature coefficients of the displacements of the two or more measurement positions different from the reference position on the surface of the at least one distance measuring device with respect to the reference position produced by measuring the distance between the objective lens incorporated in the confocal optical system and the reference position on the surface of the at least one distance measuring device set in the sample carrier and the distance between the objective lens and two or more measurement positions different from the reference position on the surface of the at least one distance measuring device at two or more different temperatures from each other, and calculating displacements of the two or more measurement positions different from the reference position on the surface of the at least one distance measuring device set in the sample carrier with respect to the reference position on the surface of the at least one distance measuring device and stored in the non-volatile memory, reading from the non-volatile memory an average temperature in the scanner when the focus position data were produced stored in the non-volatile memory, correcting the focus position data of the confocal optical system according to difference in temperature between the temperature in the scanner and the average temperature in the scanner when the focus position data of the confocal optical system were produced and in accordance with the temperature coefficients of the displacements of the two or more measurement positions different from the reference position on the surface of the at least one distance measuring device with respect to the reference position, and adjusting the position of the objective lens of the confocal optical system based on the thus corrected focus position data of the confocal optical system.
According to this preferred aspect of the present invention, the focus of the confocal optical system can be adjusted in a desired manner even when temperature in the scanner changes because the method for producing digital data of a scanner having a confocal optical system further comprises steps of reading from the non-volatile memory temperature coefficients of the displacements of the two or more measurement positions different from the reference position on the surface of the at least one distance measuring device with respect to the reference position produced by measuring the distance between the objective lens incorporated in the confocal optical system and the reference position on the surface of the at least one distance measuring device set in the sample carrier and the distance between the objective lens and two or more measurement positions different from the reference position on the surface of the at least one distance measuring device at two or more different temperatures from each other, and calculating displacements of the two or more measurement positions different from the reference position on the surface of the at least one distance measuring device set in the sample carrier with respect to the reference position on the surface of the at least one distance measuring device and stored in the non-volatile memory, reading from the non-volatile memory an average temperature in the scanner when the focus position data were produced stored in the non-volatile memory, correcting the focus position data of the confocal optical system according to difference in temperature between the temperature in the scanner and the average temperature in the scanner when the focus position data of the confocal optical system were produced and in accordance with the temperature coefficients of the displacements of the two or more measurement positions different from the reference position on the surface of the at least one distance measuring device with respect to the reference position, and adjusting the position of the objective lens of the confocal optical system based on the thus corrected focus position data of the confocal optical system.
In a further preferred aspect of the present invention, the sample carrier is constituted so as to carry two or more distance measuring devices, and the method for producing digital data of a scanner having a confocal optical system further comprises steps of reading from the non-volatile memory the position data produced by placing the sample carrier carrying two or more samples on the sample stage, measuring distances between the objective lens incorporated in the confocal optical system and three or more measurement positions different from each other on each of the two or more distance measuring devices, determining one of the measurement positions on one of the two or more distance measuring devices as the reference position, calculating displacements of the measurement positions on each of the two or more distance measuring devices with respect to the reference position, and averaging the displacements of the measurement positions on each of the two or more distance measuring devices and stored in the non-volatile memory, correcting the focus position data of the confocal optical system based on the position data for each of samples set at positions corresponding to positions of the two or more distance measuring devices set in the sample carrier, and moving the objective lens of the confocal optical system in accordance with the thus corrected focus position data of the confocal optical system, thereby adjusting the position of the objective lens.
According to this preferred aspect of the present invention, the focus of the confocal system can be accurately adjusted in a desired manner for each of the plurality of samples set in the sample carrier because the sample carrier is constituted so as to carry two or more distance measuring devices, and the method for producing digital data of a scanner having a confocal optical system further comprises steps of reading from the non-volatile memory the position data produced by placing the sample carrier carrying two or more samples on the sample stage, measuring distances between the objective lens incorporated in the confocal optical system and three or more measurement positions different from each other on each of the two or more distance measuring devices, determining one of the measurement positions on one of the two or more distance measuring devices as the reference position, calculating displacements of the measurement positions on each of the two or more distance measuring devices with respect to the reference position, and averaging the displacements of the measurement positions on each of the two or more distance measuring devices and stored in the non-volatile memory, correcting the focus position data of the confocal optical system based on the position data for each of samples set at positions corresponding to positions of the two or more distance measuring devices set in the sample carrier, and moving the objective lens of the confocal optical system in accordance with the thus corrected focus position data of the confocal optical system, thereby adjusting the position of the objective lens.
In a further preferred aspect of the present invention, the method for producing digital data of a scanner having a confocal optical system further comprises steps of reading from the non-volatile memory the temperature coefficients of displacements of the measurement positions with respect to the reference position produced by measuring distances between the objective lens incorporated in the confocal optical system and the three or more measurement positions on each of the two or more distance measuring devices with respect to the reference position determined by one of the measurement positions on one of the two or more distance measuring devices at two or more temperatures different from each other, and calculating displacements of the measurement positions on each of the two or more distance measuring devices with respect to the reference position and stored in the non-volatile memory, reading from the non-volatile memory the average temperature in the scanner when the focus position data of the confocal optical system were produced stored in the non-volatile memory, correcting the focus position data of the confocal optical system for each of the two or more distance measuring devices according to difference in temperature between the temperature in the scanner and the average temperature in the scanner when the focus position data of the confocal optical system were produced and in accordance with the temperature coefficients of the two or more distance measuring devices stored in the non-volatile memory, and moving the objective lens of the confocal optical system in accordance with the thus corrected focus position data of the confocal optical system, thereby adjusting the position of the objective lens.
According to this preferred aspect of the present invention, the focus of the confocal system can be accurately adjusted for each of the plurality of samples set in the sample carrier even when the temperature in the scanner changes because the method for producing digital data of a scanner having a confocal optical system further comprises steps of reading from the non-volatile memory the temperature coefficients of displacements of the measurement positions with respect to the reference position produced by measuring distances between the objective lens incorporated in the confocal optical system and the three or more measurement positions on each of the two or more distance measuring devices with respect to the reference position determined by one of the measurement positions on one of the two or more distance measuring devices at two or more temperatures different from each other, and calculating displacements of the measurement positions on each of the two or more distance measuring devices with respect to the reference position and stored in the non-volatile memory, reading from the non-volatile memory the average temperature in the scanner when the focus position data of the confocal optical system were produced stored in the non-volatile memory, correcting the focus position data of the confocal optical system for each of the two or more distance measuring devices according to difference in temperature between the temperature in the scanner and the average temperature in the scanner when the focus position data of the confocal optical system were produced and in accordance with the temperature coefficients of the two or more distance measuring devices stored in the non-volatile memory, and moving the objective lens of the confocal optical system in accordance with the thus corrected focus position data of the confocal optical system, thereby adjusting the position of the objective lens.
In a further preferred aspect of the present invention, the method for producing digital data of a scanner having a confocal optical system further comprises steps of reading the position data produced by measuring distances between the objective lens incorporated in the confocal optical system and at least two measurement positions different from each other in each main scanning line on each of the two or more distance measuring devices set in the sample carrier, determining one of the measurement positions on one of the two or more distance measuring devices as the reference position, and calculating displacements of the measurement positions on each of the two or more distance measuring devices with respect to the reference position, averaging the displacements of the measurement positions on each of the two or more distance measuring devices and stored in the non-volatile memory, correcting the focus position data of the confocal optical system read from the non-volatile memory based on the position data for each of the samples set at positions corresponding to positions of the two or more distance measuring devices set in the sample carrier, and moving the objective lens of the confocal optical system in accordance with the thus corrected focus position data of the confocal optical system, thereby adjusting the position of the objective lens.
In a further preferred aspect of the present invention, the method for producing digital data of a scanner having a confocal optical system further comprises steps of reading from the non-volatile memory an average value of the temperature coefficients of displacements of the measurement positions on each of the two or more distance measuring devices with respect to the reference position produced by measuring distances between the objective lens incorporated in the confocal optical system and the at least two measurement positions in each main scanning line on each of the two or more distance measuring devices at two or more temperatures different from each other, determining one of the measurement positions on one of the two or more distance measuring devices as the reference position, calculating displacements of the measurement positions on each of the two or more distance measuring devices with respect to the reference position to produce the temperature coefficients of displacements of the measurement positions with respect to the reference position, and averaging the temperature coefficients and stored in the non-volatile memory, reading from the non-volatile memory the average temperature in the scanner when the focus position data were produced stored in the non-volatile memory, correcting the focus position data of the confocal optical system read from the non-volatile memory for each of the two or more distance measuring devices according to difference in temperature between the temperature in the scanner and the average temperature in the scanner when the focus position data of the confocal optical system were produced and in accordance with the temperature coefficients of the two or more distance measuring devices, and moving the objective lens of the confocal optical system in accordance with the thus corrected focus position data of the confocal optical system, thereby adjusting the position of the objective lens.
According to this preferred aspect of the present invention, the focus of the confocal system can be more accurately adjusted for each of the plurality of samples set in the sample carrier even when the temperature in the scanner changes because the method for producing digital data of a scanner having a confocal optical system further comprises steps of reading from the non-volatile memory an average value of the temperature coefficients of displacements of the measurement positions on each of the two or more distance measuring devices with respect to the reference position produced by measuring distances between the objective lens incorporated in the confocal optical system and the at least two measurement positions in each main scanning line on each of the two or more distance measuring devices at two or more temperatures different from each other, determining one of the measurement positions on one of the two or more distance measuring devices as the reference position, calculating displacements of the measurement positions on each of the two or more distance measuring devices with respect to the reference position to produce the temperature coefficients of displacements of the measurement positions with respect to the reference position, and averaging the temperature coefficients and stored in the non-volatile memory, reading from the non-volatile memory the average temperature in the scanner when the focus position data were produced stored in the non-volatile memory, correcting the focus position data of the confocal optical system read from the non-volatile memory for each of the two or more distance measuring devices according to difference in temperature between the temperature in the scanner and the average temperature in the scanner when the focus position data of the confocal optical system were produced and in accordance with the temperature coefficients of the two or more distance measuring devices, and moving the objective lens of the confocal optical system in accordance with the thus corrected focus position data of the confocal optical system, thereby adjusting the position of the objective lens.
In a further preferred aspect of the present invention, the method for producing digital data of a scanner having a confocal optical system further comprises steps of reading the position data produced by measuring distances between the objective lens incorporated in the confocal optical system and three measurement positions different from each other in each main scanning line on each of the two or more distance measuring devices set in the sample carrier, determining one of the measurement positions on one of the two or more distance measuring devices as the reference position, calculating displacements of the measurement positions on each of the two or more distance measuring devices with respect to the reference position, and averaging the displacements of the measurement positions on each of the two or more distance measuring devices and stored in the non-volatile memory, correcting the focus position data of the confocal optical system read from the non-volatile memory based on the position data for each of the samples set at positions corresponding to positions of the two or more distance measuring devices set in the sample carrier, and moving the objective lens of the confocal optical system in accordance with the thus corrected focus position data of the confocal optical system, thereby adjusting the position of the objective lens.
In a further preferred aspect of the present invention, the method for producing digital data of a scanner having a confocal optical system further comprises steps of reading from the non-volatile memory an average value of the temperature coefficients of displacements of the measurement positions on each of the two or more distance measuring devices with respect to the reference position produced by measuring distances between the objective lens incorporated in the confocal optical system and the three measurement positions in each main scanning line on each of the two or more distance measuring devices at two or more temperatures different from each other, determining one of the measurement positions on one of the two or more distance measuring devices as the reference position, calculating displacements of the measurement positions on each of the two or more distance measuring devices with respect to the reference position to produce the temperature coefficients of displacements of the measurement positions with respect to the reference position, and averaging the temperature coefficients and stored in the non-volatile memory as temperature coefficients of the two or more distance measuring devices, reading from the non-volatile memory the average temperature in the scanner when the focus position data were produced stored in the non-volatile memory, correcting the focus position data of the confocal optical system read from the non-volatile memory for each of the two or more distance measuring devices according to difference in temperature between the temperature in the scanner and the average temperature in the scanner when the focus position data of the confocal optical system were produced and in accordance with the temperature coefficients of the two or more distance measuring devices, and moving the objective lens of the confocal optical system in accordance with the thus corrected focus position data of the confocal optical system, thereby adjusting the position of the objective lens.
In a further preferred aspect of the present invention, the method for producing digital data of a scanner having a confocal optical system further comprises steps of reading from the non-volatile memory the position data produced by averaging displacements of the measurement positions with respect to the reference position for every predetermined number of the main scanning lines on each of the two or more distance measuring devices, correcting the focus position data of the confocal optical system read from the non-volatile memory based on the position data for every predetermined number of the main scanning lines corresponding to the predetermined number of the main scanning lines on each of the samples set at positions in the sample carrier corresponding to positions where the two or more distance measuring devices are set, and moving the objective lens of the confocal optical system in accordance with the thus corrected focus position data of the confocal optical system, thereby adjusting the position of the objective lens.
According to this preferred aspect of the present invention, the position of the objective lens can be more precisely adjusted and the focus of the confocal system can therefore be more accurately adjusted for each of the plurality of samples set in the sample carrier, because the method for producing digital data of a scanner having a confocal optical system further comprises steps of reading from the non-volatile memory the position data produced by averaging displacements of the measurement positions with respect to the reference position for every predetermined number of the main scanning lines on each of the two or more distance measuring devices, correcting the focus position data of the confocal optical system read from the non-volatile memory based on the position data for every predetermined number of the main scanning lines corresponding to the predetermined number of the main scanning lines on each of the samples set at positions in the sample carrier corresponding to positions where the two or more distance measuring devices are set, and moving the objective lens of the confocal optical system in accordance with the thus corrected focus position data of the confocal optical system, thereby adjusting the position of the objective lens.
In a further preferred aspect of the present invention, the method for producing digital data of a scanner having a confocal optical system further comprises steps of reading from the non-volatile memory an average value of the temperature coefficients of displacements of the measurement positions on each of the two or more distance measuring devices with respect to the reference position produced by measuring distances between the objective lens incorporated in the confocal optical system and three measurement positions in each main scanning line for each predetermined number of main scanning lines on each of the two or more distance measuring devices at two or more temperatures different from each other, determining one of the measurement positions on one of the two or more distance measuring devices as the reference position, calculating displacements of the measurement positions with respect to the reference position for every predetermined number of the main scanning lines on each of the two or more distance measuring devices to produce the temperature coefficients of displacements of the measurement positions with respect to the reference position, averaging the temperature coefficients of displacements of the measurement positions with respect to the reference position and stored in the non-volatile memory as temperature coefficients for every predetermined number of the main scanning lines on each of the two or more distance measuring devices, reading from the non-volatile memory the average temperature in the scanner when the focus position data were produced stored in the non-volatile memory, correcting the focus position data of the confocal optical system read from the non-volatile memory according to difference in temperature between the temperature in the scanner and the average temperature in the scanner when the focus position data of the confocal optical system were produced and in accordance with the temperature coefficients for every predetermined number of the main scanning lines on each of the two or more distance measuring devices for every predetermined number of the main scanning lines corresponding to the predetermined number of the main scanning lines on each of the samples set at positions in the sample carrier corresponding to positions where the two or more distance measuring devices are set, and moving the objective lens of the confocal optical system in accordance with the thus corrected focus position data of the confocal optical system, thereby adjusting the position of the objective lens.
According to this preferred aspect of the present invention, the method for producing digital data of a scanner having a confocal optical system further comprises steps of reading from the non-volatile memory an average value of the temperature coefficients of displacements of the measurement positions on each of the two or more distance measuring devices with respect to the reference position produced by measuring distances between the objective lens incorporated in the confocal optical system and three measurement positions in each main scanning line for each predetermined number of main scanning lines on each of the two or more distance measuring devices at two or more temperatures different from each other, determining one of the measurement positions on one of the two or more distance measuring devices as the reference position, calculating displacements of the measurement positions with respect to the reference position for every predetermined number of the main scanning lines on each of the two or more distance measuring devices to produce the temperature coefficients of displacements of the measurement positions with respect to the reference position, averaging the temperature coefficients of displacements of the measurement positions with respect to the reference position and stored in the non-volatile memory as temperature coefficients for every predetermined number of the main scanning lines on each of the two or more distance measuring devices, reading from the non-volatile memory the average temperature in the scanner when the focus position data were produced stored in the non-volatile memory, correcting the focus position data of the confocal optical system read from the non-volatile memory according to difference in temperature between the temperature in the scanner and the average temperature in the scanner when the focus position data of the confocal optical system were produced and in accordance with the temperature coefficients for every predetermined number of the main scanning lines on each of the two or more distance measuring devices for every predetermined number of the main scanning lines corresponding to the predetermined number of the main scanning lines on each of the samples set at positions in the sample carrier corresponding to positions where the two or more distance measuring devices are set, and moving the objective lens of the confocal optical system in accordance with the thus corrected focus position data of the confocal optical system, thereby adjusting the position of the objective lens, and, therefore, the position of the objective lens can be more precisely adjusted even when the temperature in the scanner changes so that it is possible to more accurately adjust the focus of the confocal system for each of the plurality of samples set in the sample carrier.
In a further preferred aspect of the present invention, the objective lens of the confocal optical system is adjusted by a stepping motor.
In a further preferred aspect of the present invention, the method for producing digital data of a scanner having a confocal optical system further comprises steps of reading from the non-volatile memory coefficients of an nth order function produced by plotting integrated values of signal intensity of fluorescence emission or photoluminescence emission and fitting the plotted values with the nth order function and stored in the non-volatile memory, reading from the non-volatile memory displacements of measurement positions on the at least one distance measuring device with respect to the reference position stored in the non-volatile memory, producing shading correction data for correcting shading of digital data of the at least one sample based on the coefficients of the nth order function and the displacements of measurement positions on the at least one distance measuring device with respect to the reference position, and correcting digital data of the at least one sample based on the shading correction data.
According to this preferred aspect of the present invention, it is possible to produce the shading correction data as well as the focus position data of the confocal optical system and produce the digital data of the at least one sample in which the shading has been corrected, because the method for producing digital data of a scanner having a confocal optical system further comprises steps of reading from the non-volatile memory coefficients of an nth order function produced by plotting integrated values of signal intensity of fluorescence emission or photoluminescence emission and fitting the plotted values with the nth order function and stored in the non-volatile memory, reading from the non-volatile memory displacements of measurement positions on the at least one distance measuring device with respect to the reference position stored in the non-volatile memory, producing shading correction data for correcting shading of digital data of the at least one sample based on the coefficients of the nth order function and the displacements of measurement positions on the at least one distance measuring device with respect to the reference position, and correcting digital data of the at least one sample based on the shading correction data.
In a further preferred aspect of the present invention, the method for producing digital data of a scanner having a confocal optical system further comprises steps of setting a shading estimation device in which a mask of metal is formed on a support having a property to release fluorescence emission upon being irradiated with a laser beam and capable of being processed while retaining optical flatness, thereby regularly forming a plurality of openings through which the support is exposed in the sample carrier, placing the sample carrier on the sample stage, scanning the shading estimation device with the laser beam via the openings to stimulate the support at the plurality of openings, photoelectrically detecting fluorescence emission released from the support via the plurality of openings to produce analog data, digitizing the analog data, producing digital data of the shading estimation device based on the thus produced digital data, producing shading correction data based on the digital data of the shading estimation device, storing the thus produced shading correction data in the non-volatile memory or the memory of the scanner, and reading the shading correction data to correct digital data of the at least one samples therewith.
According to this preferred aspect of the present invention, since it is possible to correct digital data of the at least one samples using the shading correction data read from the non-volatile memory or the memory of the scanner because the method for producing digital data of a scanner having a confocal optical system further comprises steps of setting a shading estimation device in which a mask of metal is formed on a support having a property to release fluorescence emission upon being irradiated with a laser beam and capable of being processed while retaining optical flatness, thereby regularly forming a plurality of openings through which the support is exposed in the sample carrier, placing the sample carrier on the sample stage, scanning the shading estimation device with the laser beam via the openings to stimulate the support at the plurality of openings, photoelectrically detecting fluorescence emission released from the support via the plurality of openings to produce analog data, digitizing the analog data, producing digital data of the shading estimation device based on the thus produced digital data, producing shading correction data based on the digital data of the shading estimation device, storing the thus produced shading correction data in the non-volatile memory or the memory of the scanner, and reading the shading correction data to correct digital data of the at least one samples therewith.
In a further preferred aspect of the present invention, each of the plurality of openings is constituted as a slit.
In a further preferred aspect of the present invention, each of the plurality of openings is constituted as a pinhole.
In a further preferred aspect of the present invention, the method for producing digital data of a scanner having a confocal optical system further comprises steps of adjusting a focus of the confocal optical system to the opening located at the reference position among the plurality of openings regularly formed in the shading estimation device, scanning the shading estimation device with the laser beam via the plurality of openings, stimulating the support via the plurality of openings, photoelectrically detecting fluorescence emission released from the support to produce analog data, digitizing the analog data to produce digital data of the shading estimation device, producing the shading correction data based on the digital data of the shading estimation device, and storing the thus produced shading correction data in the non-volatile memory or the memory of the scanner.
According to this preferred aspect of the present invention, since the focus of the confocal optical system is adjusted to the opening located at the reference position in an optimum manner, the signal intensity of digital data produced by stimulating the support at openings other than the opening located at the reference position and photoelectrically detecting fluorescence emission released from the support is lower than the signal intensity of digital data produced by stimulating the support at the opening located at the reference position and photoelectrically detecting fluorescence emission released from the support. Therefore, the shading of the digital data of the at least one sample can be effectively corrected by producing shading correction data which can correct the digital data so that the signal intensity of digital data produced by stimulating the support at openings other than the opening located at the reference position and photoelectrically detecting fluorescence emission released from the support becomes equal to the signal intensity of digital data produced by stimulating the support at the opening located at the reference position and photoelectrically detecting fluorescence emission released from the support, correcting the digital data of the at least one sample using the thus produced shading correction data.
In a further preferred aspect of the present invention, the support is formed of a material selected from a group consisting of IV group elements, II-VI group compounds, III-V group compounds and complexes thereof.
In a further preferred aspect of the present invention, the mask of metal is formed of a material selected from a group consisting of chromium, aluminum, gold, nickel-chromium alloy and titanium-nickel-chromium alloy.
The above and other objects and features of the present invention will become apparent from the following description made with reference to the accompanying drawings.